Phenothiazine diaminium salts and their use

ABSTRACT

The invention relates to compounds of general formula (I): wherein: each of R 1  and R 9  is independently selected from: —H, C 1-4 alkyl, C 2-4 alkenyl, and halogenated C 1-4 alkyl; each of R 3NA  and R 3NB  is independently selected from: —H, C 1-4 alkyl, C 2-4 alkenyl, and halogenated C 1-4 alkyl; each of R7NA and R7NB is independently selected from: —H, C 1-4 alkyl, C 2-4 alkenyl, and halogenated C 1-4 alkyl; and wherein: each of R A  and R B  is independently selected from: C 1-4 alkyl, halogenated C 1-4 alkyl, and C 6-10 aryl; or R A  and R B  are linked to form a group selected from: C 1-6  alkylene and C 6-10  arylene; and pharmaceutically acceptable salts thereof, which are useful in the treatment of, for example, Alzheimer&#39;s disease. In other aspects the invention also relates to novel formulations of 3,7-diamino-10H-phenothiazinium salts.

TECHNICAL FIELD

This invention pertains generally to the field of phenothiazinecompounds, in particular certain phenothiazine diaminium salts,including uses and formulations thereof. In some embodiments theinvention relates to bis(sulfonic acid) salts of diaminophenothiazinecompounds such as N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine.The compounds of the invention are useful, for example, in the treatmentof tauopathies such as Alzheimer's disease (AD).

BACKGROUND

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Any sub-titles herein are included for convenience only, and are not tobe construed as limiting the disclosure in any way.

Conditions of dementia are frequently characterised by a progressiveaccumulation of intracellular and/or extracellular deposits ofproteinaceous structures such as β-amyloid plaques and neurofibrillarytangles (NFTs) in the brains of affected patients. The appearance ofthese lesions largely correlates with pathological neurofibrillarydegeneration and brain atrophy, as well as with cognitive impairment(see, e.g., Mukaetova-Ladinska, E. B. et al., 2000, Am. J. Pathol., Vol.157, No. 2, pp. 623-636).

In Alzheimer's disease, both neuritic plaques and NFTs contain pairedhelical filaments (PHFs), of which a major constituent is themicrotubule-associated protein tau (see, e.g., Wischik et al., 1988,PNAS USA, Vol. 85, pp. 4506-4510). Plaques also contain extracellularβ-amyloid fibrils derived from the abnormal processing of amyloidprecursor protein (APP) (see, e.g., Kang et al., 1987, Nature, Vol. 325,p. 733). An article by Wischik et al. (in ‘Neurobiology of Alzheimer'sDisease’, 2nd Edition, 2000, Eds. Dawbarn, D. and Allen, S. J., TheMolecular and Cellular Neurobiology Series, Bios Scientific Publishers,Oxford) discusses in detail the putative role of tau protein in thepathogenesis of neurodegenerative dementias. Loss of the normal form oftau, accumulation of pathological PHFs, and loss of synapses in themid-frontal cortex all correlate with associated cognitive impairment.Furthermore, loss of synapses and loss of pyramidal cells both correlatewith morphometric measures of tau-reactive neurofibrillary pathology,which parallels, at a molecular level, an almost total redistribution ofthe tau protein pool from a soluble to a polymerised form (i.e., PHFs)in Alzheimer's disease.

Tau exists in alternatively-spliced isoforms, which contain three orfour copies of a repeat sequence corresponding to themicrotubule-binding domain (see, e.g., Goedert, M., et al., 1989, EMBOJ., Vol. 8, pp. 393-399; Goedert, M., et al., 1989, Neuron, Vol. 3, pp.519-526). Tau in PHFs is proteolytically processed to a core domain(see, e.g., Wischik, C. M., et al., 1988, PNAS USA, Vol. 85, pp.4884-4888; Wischik et al., 1988, PNAS USA, Vol. 85, pp. 4506-4510;Novak, M., et al., 1993, EMBO J., Vol. 12, pp. 365-370) which iscomposed of a phase-shifted version of the repeat domain; only threerepeats are involved in the stable tau-tau interaction (see, e.g.,Jakes, R., et al., 1991, EMBO J., Vol. 10, pp. 2725-2729). Once formed,PHF-like tau aggregates act as seeds for the further capture and providea template for proteolytic processing of full-length tau protein (see,e.g., Wischik et al., 1996, PNAS USA, Vol. 93, pp. 11213-11218).

The phase shift which is observed in the repeat domain of tauincorporated into PHFs suggests that the repeat domain undergoes aninduced conformational change during incorporation into the filament.During the onset of AD, it is envisaged that this conformational changecould be initiated by the binding of tau to a pathological substrate,such as damaged or mutated membrane proteins (see, e.g., Wischik, C. M.,et al., 1997, in “Microtubule-associated proteins: modifications indisease”, Eds. Avila, J., Brandt, R. and Kosik, K. S. (Harwood AcademicPublishers, Amsterdam) pp. 185-241).

In the course of their formation and accumulation, PHFs first assembleto form amorphous aggregates within the cytoplasm, probably from earlytau oligomers which become truncated prior to, or in the course of, PHFassembly (see, e.g., Mena, R., et al., 1995, Acta Neuropathol., Vol. 89,pp. 50-56; Mena, R., et al., 1996, Acta Neuropathol., Vol. 91, pp.633-641). These filaments then go on to form classical intracellularNFTs. In this state, the PHFs consist of a core of truncated tau and afuzzy outer coat containing full-length tau (see, e.g., Wischik et al.,1996, PNAS USA, Vol. 93, pp. 11213-11218). The assembly process isexponential, consuming the cellular pool of normal functional tau andinducing new tau synthesis to make up the deficit (see, e.g., Lai, R. Y.K., et al., 1995, Neurobiology of Ageing, Vol. 16, No. 3, pp. 433-445).Eventually, functional impairment of the neurone progresses to the pointof cell death, leaving behind an extracellular NFT. Cell death is highlycorrelated with the number of extracellular NFTs (see, e.g., Wischik etal., in ‘Neurobiology of Alzheimer's Disease’, 2nd Edition, 2000, Eds.Dawbarn, D. and Allen, S. J., The Molecular and Cellular NeurobiologySeries, Bios Scientific Publishers, Oxford). As tangles are extrudedinto the extracellular space, there is progressive loss of the fuzzyouter coat of the neurone with corresponding loss of N-terminal tauimmunoreactivity, but preservation of tau immunoreactivity associatedwith the PHF core (see, e.g., Bondareff, W. et al., 1994, J. Neuropath.Exper. Neurol., Vol. 53, No. 2, pp. 158-164).

Diaminophenothiazine Compounds

Methythioninium Chloride (MTC) (also known as Methylene blue (MB);methylthionine chloride; tetramethylthionine chloride;3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9;tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathioniumchloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet;and Urolene Blue®) is a low molecular weight (319.86), water soluble,tricyclic organic compound of the following formula:

Methythioninium Chloride (MTC) is a well known phenothiazine dye andredox indicator and has also been used as an optical probe ofbiophysical systems, as an intercalator in nanoporous materials, as aredox mediator, and in photoelectrochromic imaging. Methythioniniumchloride (MTC) and other diaminophenothiazines have been described asinhibitors of protein aggregation in diseases in which proteinsaggregate pathologically.

In particular, diaminopenothiazines including MTC have been shown toinhibit tau protein aggregation and to disrupt the structure of PHFs,and reverse the proteolytic stability of the PHF core (see, e.g., WO96/30766, Hofmann-La Roche). Such compounds were disclosed for use inthe treatment or prophylaxis of various diseases, including Alzheimer'sdisease.

WO2007/110630 (WisTa Laboratories Ltd) also discloses certain specificdiaminophenothiazine compounds related to MTC, including ETC, DEMTC,DMETC, DEETC, MTZ, ETZ, MTI, MTILHI, ETI, ETLHI, MTN, and ETN, which areuseful as drugs, for example in the treatment of Alzheimer's disease.

Additionally, WO 2005/030676 (The University Court of the University ofAberdeen) discusses radiolabelled phenothiazines, and their use indiagnosis and therapy, for example, of tauopathies.

Methythioninium chloride (MTC) has also been disclosed for other medicaluses. For example it is currently used to treat methemoglobinemia (acondition that occurs when the blood cannot deliver oxygen where it isneeded in the body). MTC is also used as a medical dye (for example, tostain certain parts of the body before or during surgery); a diagnostic(for example, as an indicator dye to detect certain compounds present inurine); a mild urinary antiseptic; a stimulant to mucous surfaces; atreatment and preventative for kidney stones; and in the diagnosis andtreatment of melanoma.

MTC has been used to treat malaria, either singly (see, e.g., Guttmann,P. and Ehrlich, P., 1891, “Uber die wirkung des methylenblau beimalaria,” Berl. Klin. Woschenr., Vol. 28, pp. 953-956) or in combinationwith chloroquine (see, e.g., Schirmer, H., et al., 2003, “Methylene blueas an antimalarial agent,” Redox Report, Vol. 8, pp. 272-275;Rengelshausen, J., et al., 2004, “Pharmacokinetic interaction ofchloroquine and methylene blue combination against malaria,” EuropeanJournal of Clinical Pharmacology, Vol. 60, pp. 709-715).

MTC (under the name Virostat®, from Bioenvision Inc., New York) has alsoshown potent viricidal activity in vitro. Specifically Virostat® iseffective against viruses such as HIV and West Nile Virus in laboratorytests. Virostat® is also currently in clinical trials for the treatmentof chronic Hepatitis C, a viral infection of the liver. The virus, HCV,is a major cause of acute hepatitis and chronic liver disease, includingcirrhosis and liver cancer. MTC, when combined with light, can alsoprevent the replication of nucleic acid (DNA or RNA). Plasma, plateletsand red blood cells do not contain nuclear DNA or RNA. When MTC isintroduced into the blood components, it crosses bacterial cell walls orviral membrane then moves into the interior of the nucleic acidstructure. When activated with light, the compound then binds to thenucleic acid of the viral or bacterial pathogen, preventing replicationof the DNA or RNA. Because MTC can inactivate pathogens, it has thepotential to reduce the risk of transmission of pathogens that wouldremain undetected by testing.

Oral and parenteral formulations of MTC have been commercially availablein the United States, usually under the name Urolene Blue®.

Reduced (‘Leuco’) Forms

MTC, a phenothiazin-5-ium salt, may be considered to be an “oxidizedform” in relation to the corresponding 10H-phenothiazine compound,N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine, which may beconsidered to be a “reduced form”:

The “reduced form” (or “leuco form”) is known to be unstable and can bereadily and rapidly oxidized to give the corresponding “oxidized” form.

May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398)have shown that human erythrocytes sequentially reduce and take up MTC;that MTC itself is not taken up by the cells; that it is the reducedform of MTC that crosses the cell membrane; that the rate of uptake isenzyme dependent; and that both MTC and reduced MTC are concentrated incells (reduced MTC re-equilibrates once inside the cell to form MTC).

MTC and similar drugs are taken up in the gut and enter the bloodstream.Unabsorbed drug percolates down the alimentary canal, to the distal gut.One important undesired side-effect is the effect of the unabsorbed drugin the distal gut, for example, sensitisation of the distal gut and/orantimicrobial effects of the unabsorbed drug on flora in the distal gut,both leading to diarrhoea. Therefore, it is desirable to minimize theamount of drug that percolates to the distal gut. By increasing thedrug's uptake in the gut (i.e., by increasing the drug'sbioavailability), dosage may be reduced, and the undesired side-effects,such as diarrhoea, may be ameliorated.

Since it is the reduced form of MTC that is taken up by cells, it may bedesirable to administer the reduced form to patients. This may alsoreduce reliance on the rate limiting step of enzymatic reduction.

WO 02/055720 (The University Court of the University of Aberdeen)discloses the use of reduced forms of certain diaminophenothiazines forthe treatment of protein aggregating diseases, primarily tauopathies.

WO02007/110627 (WisTa Laboratories Ltd) disclosed certain3,7-diamino-10H-phenothiazinium salts, effective as drugs or pro-drugsfor the treatment of diseases including Alzheimer's disease. Thesecompounds are also in the “reduced” or “leuco” form when considered inrespect of MTC. These included the following salts:

N,N,N′,N′-tetramethyl-10H-phenothiazine- 3,7-diaminium di(chloride),(LMT•2HCl)

N,N,N′,N′-tetramethyl-10H-phenothiazine- 3,7-diaminium di(bromide),(LMT•2HBr)

N,N,N′,N′-tetramethyl-10H-phenothiazine- 3,7-diaminium di(iodide),(LMT•2HI)

Although providing certain advantages over the use of MTC, the synthesisof LMT.2HCl under certain conditions may result in CH₃Cl being trappedwithin the crystal. This then needs to be removed since CH₃Cl is toxicand levels need to be kept below safety levels.

Furthermore LMT.2HBr contains bromide ions. This is in principle lessdesirable since bromide is toxic either at high levels or with chronicdosing and, at lower levels, can causes side effects such as confusionin patients.

Therefore it can be seen the provision of further salts ofmethylthioninium compounds, having one or more desirable properties overthose already known, would be a contribution to the art.

Furthermore the provision of novel formulations of methylthioniniumcompounds which enhance stability, absorption, and\or otherwise improvetheir effectiveness as therapeutics would be a contribution to the art.

SUMMARY OF THE INVENTION

The present inventors have now identified a new class of stablephenothiazine diaminium compounds which have improved properties ascompared to previously disclosed diaminophenothiazine compounds andsalts.

The properties of the compounds are described hereinafter, whereby itcan be seen that in preferred embodiments the invention can provide oneor more of improved physical, pharmacokinetic, biochemical or otherbeneficial properties.

In other aspects the present inventors also provide novel formulationsof 3,7-diamino-10H-phenothiazinium salts.

In one aspect the present invention provides certain compounds,specifically, certain phenothiazine diaminium compounds, as describedherein.

The compound may be selected from compounds of general formula (I):

wherein:

-   -   each of R¹ and R⁹ is independently selected from: —H, C₁₋₄alkyl,        C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(3NA) and R^(3NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(7NA) and R^(7NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;        and wherein:    -   each of R^(A) and R^(B) is independently selected from:    -   C₁₋₄alkyl, halogenated C₁₋₄alkyl, and C₆₋₁₀aryl;    -   or    -   R^(A) and R^(B) are linked to form a group R^(AB), wherein        R^(AB) is selected from:    -   C₁₋₆ alkylene and C₆₋₁₀ arylene;        and pharmaceutically acceptable salts thereof.

Another aspect of the invention pertains to processes for synthesizing acompound as described above.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising a compound as described herein and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the invention pertains to a method of preparing apharmaceutical composition comprising admixing a compound as describedherein and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention pertains to a pharmaceutical compositionin solid dosage form, comprising a compound as described herein andfurther comprising at least one diluent suitable for dry compression,and optionally one or more other excipients.

Another aspect of the invention pertains to a process for themanufacture of a pharmaceutical composition by a dry compression method,said composition being a solid dosage form comprising a compound asdescribed herein, at least one diluent suitable for dry compression, andoptionally one or more other excipients.

Another aspect of the invention pertains to a free-flowing, cohesivepowder, comprising a compound as described herein and at least onediluent suitable for dry compression, and optionally one or more otherexcipients, said powder being capable of being compressed into a soliddosage form.

Another aspect of the present invention pertains to a method ofreversing and/or inhibiting the aggregation of a protein (e.g., a tauprotein, a synuclein, etc.), for example, aggregation of a proteinassociated with a neurodegenerative disease and/or clinical dementia,comprising contacting the protein with an effective amount of a compoundor composition as described herein. Such a method may be performed invitro, or in vivo.

Another aspect of the present invention pertains to a method oftreatment or prophylaxis of a disease condition in a subject comprisingadministering to said subject a prophylactically or therapeuticallyeffective amount of a compound as described herein, preferably in theform of a pharmaceutical composition, preferably a pharmaceuticalcomposition in solid dosage form, as further described herein.

Another aspect of the present invention pertains to a compound orcomposition as described herein for use in a method of treatment orprophylaxis (e.g., of a disease condition) of the human or animal bodyby therapy.

Another aspect of the present invention pertains to use of a compound orcomposition as described herein, in the manufacture of a medicament foruse in the treatment or prophylaxis of a disease condition.

In some embodiments, the disease condition is a disease of proteinaggregation.

In some embodiments, the disease condition is a tauopathy, e.g., aneurodegenerative tauopathy, e.g., Alzheimer's disease or other diseasedescribed hereinafter.

In some embodiments, the disease condition is skin cancer, e.g.,melanoma.

In some embodiments, the disease condition is a viral, bacterial orprotozoal disease condition, e.g., Hepatitis C, HIV, West Nile Virus(WNV), or malaria.

Another aspect of the present invention pertains to a method ofinactivating a pathogen in a sample (for example a blood or plasmasample), comprising the steps of introducing a compound or compositionas described herein, into the sample, and then exposing the sample tolight.

Another aspect of the present invention pertains to a kit comprising (a)a compound as described herein, preferably provided as a pharmaceuticalcomposition and in a suitable container and/or with suitable packaging;and (b) instructions for use, for example, written instructions on howto administer the compound or composition.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ¹H NMR spectrum of an exemplary compound of theinvention (LMT.2MsOH) in deuterated methanol (CD₃OD) at 600 MHz.

FIG. 2 shows the ¹³C NMR spectrum of LMT.2MsOH in CD₃OD at a frequencyof 100.56 MHz.

FIG. 3 shows the DEPT-135 spectrum of LMT.2MsOH in CD₃OD at a frequencyof 100.56 MHz.

FIG. 4 shows the HSQC spectrum of LMT.2MsOH in CD₃OD at a frequency of100.56 MHz.

FIG. 5 shows the an expanded section of the HSQC spectrum of LMT.2MsOHin CD₃OD at a frequency of 100.56 MHz.

FIG. 6 shows the infrared (FT-IR) spectrum of LMT.2MsOH (KBr).

FIG. 7 shows the electron impact (EI) mass spectrum of LMT.2MsOH.

FIG. 8 shows the electrospray ionisation (ESI) mass spectrum ofLMT.2MsOH.

FIG. 9 shows the UV/Vis is spectrum of LMT.2MsOH in de-ionised water.

FIG. 10 shows the HPLC trace for LMT.2MsOH.

FIG. 11 shows a powder X-ray diffractogram for LMT.2MsOH, measured withCu Kα radiation.

FIG. 12 shows the FT-Raman spectrum for crystalline LMT.2MsOH. The mostintense signals are found at 1615 cm⁻¹, 1588 cm⁻¹ 1258 cm⁻¹, and 1042cm⁻¹.

FIG. 13 shows the thermogravimetric profile for crystalline LMT.2MsOH. Aconstant weight was detected by TG and TG-FTIR up to the beginning ofdecomposition at 240-270° C.

FIG. 14 shows the differential scanning calorimetry analysis forcrystalline LMT.2MsOH. A sharp m.p. at 271° C. (ΔH=87 J/g) wasimmediately followed by decomposition.

FIGS. 15 a and 15 b show the dynamic vapour sorption (DVS) curve forcrystalline LMT.2MsOH measured at 25° C. with 5%/h scanning rate. Thehorizontal dashed lines indicate steps of water uptake of oneequivalent. A stable weight of the sample (less than 0.5% weight change)was observed in the relative humidity (r.h.) range between 0% and 70%.Above this r.h., the water uptake increased rapidly, and the sampleultimately deliquesced. Upon drying, the water content decreased againto approximately 4 equiv. at 50% r.h. The DVS curve of the crystallinedihydrochloride salt (LMT.2HCl) is shown for comparison as a dashedline, the DVS curve of the dihydrobromide salt (LMT.2HBr) as a dottedline.

FIG. 15 c shows the dynamic vapour sorption (DVS) curve for crystallineLMT.2MsOH as a function of time. The relative humidity is also indicated(right axis). The horizontal dashed lines indicate steps of oneequivalent water uptake.

FIG. 16 shows polarizing microscopy pictures of the LMT.2MsOH (left) andrecrystallized LMT.2MsOH (right). Crystals of up to 100 μm in size wereobtained by recrystallization from 2-PrOH/water. Crystals areirregularly shaped.

FIGS. 17 a-c shows the X-ray crystal structures of LMTEsOH, LMT.EDSA.and LMT.2MsOH

FIG. 18 shows a comparison of the plasma concentration in pig of the MTmoiety over time following dosing of LMT.2HBr, LMT.2HCl and LMT.2MsOH.

FIG. 19 is a diagram of the apparatus used in the dissolution studies(see Formulation Example 12).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a new class of phenothiazinediaminium compounds which have desirable physical or other propertiesand\or surprisingly improved activity as compared to previouslydisclosed diaminophenothiazine compounds and salts.

In other aspects they have additionally provided novel formulations ofphenothiazine diaminium compounds, including (but not limited to) theclass above.

The Compounds

In general terms, unless context demands otherwise, the compounds of theinvention may be described as bis(sulfonate) salts (or bis(sulfonicacid) salts) of 3,7-diamino-10H-phenothiazine compounds. In other words,the compounds are salts of the corresponding3,7-diamino-10H-phenothiazine compounds with organic sulfonic acids.

More specifically, a compound of the invention is a bis(sulfonate) saltof a compound of general formula:

wherein R¹, R⁹, R^(3NA), R^(3NB), R^(7NA) and R^(7NB) are as definedabove.

In some embodiments, the salt is a bis(alkylsulfonate) salt or abis(arylsulfonate) salt.

In some embodiments, the salt is selected from a bis(methanesulfonate)salt, a bis(ethanesulfonate) salt, a bis(p-toluenesulfonate) salt, abis(benzenesulfonate) salt, an ethanedisulfonate salt, apropanedisulfonate salt, or a naphthalenedisulfonate salt.

In some embodiments, the salt is a bis(methanesulfonate) salt (which mayalso be called a bis(mesylate) salt).

In some embodiments, the salt is a bis(ethanesulfonate) salt (which mayalso be called a bis(esylate) salt).

In some embodiments, the salt is a bis(p-toluenesulfonate) salt (whichmay also be called a bis(tosylate) salt).

In some embodiments, the salt is a bis(benzenesulfonate) salt.

In some embodiments, the salt is an ethanedisulfonate salt.

In some embodiments, the salt is a propanedisulfonate salt.

In some embodiments, the salt is a naphthalenedisulfonate salt,preferably a naphthalene-1,5-disulfonate salt.

In other words, the compounds of the invention can be considered to beproducts obtainable from the reaction of a 3,7-diamino-10H-phenothiazinecompound, for example as set out above, with two organic sulfonic acidmoieties (R^(A)SO₃H and R^(B)SO₃H). The two organic sulfonic acidmoieties may optionally be present on the same molecule, i.e. whereR^(A) and R^(B) are linked.

In some embodiments, compounds of the invention are selected fromcompounds of general formula (I):

wherein:

-   -   each of R¹ and R⁹ is independently selected from: —H, C₁₋₄alkyl,        C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(3NA) and R^(3NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(7NA) and R^(7NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;        and wherein:    -   each of R^(A) and R^(B) is independently selected from:    -   C₁₋₄alkyl, halogenated C₁₋₄alkyl, and C₆₋₁₀aryl;    -   or    -   R^(A) and R^(B) are linked to form a group R^(AB), wherein        R^(AB) is selected from:    -   C₁₋₆alkylene and C₆₋₁₀ arylene;        and pharmaceutically acceptable salts, solvates, and hydrates        thereof.

Compounds of the invention are represented herein by a general formulashowing the structure of the 3,7-diamino-10H-phenothiazine compound,with the 3,7-diamino groups being in protonated form.

The resultant doubly positively-charged species is associated with twosulfonate counterion moieties (which may optionally be present on thesame molecule, i.e. where R^(A) and R^(B) are linked):

However, as will be understood by one skilled in the art, the same saltcould equally be represented in other ways, such as, for example:

etc.Further Definitions And Preferences

The term “C₁₋₄ alkyl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a hydrocarbon compound havingfrom 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or acombination thereof.

Similarly, the term “C₂₋₄alkenyl” pertains to a monovalent moietyobtained by removing a hydrogen atom from a C₂₋₄ alkene compound (i.e. ahydrocarbon compound containing at least one double bond and from 2 to 4carbon atoms).

The term “C₁₋₆ alkylene”, as used herein, pertains to a bidentate moietyobtained by removing two hydrogen atoms, either both from the samecarbon atom, or one from each of two different carbon atoms, of analiphatic linear hydrocarbon compound having from 1 to 6 carbon atoms.

In some embodiments, C₁₋₄alkyl groups may be selected from: linearC₁₋₄alkyl groups, such as -Me, -Et, -nPr, -iPr, and -nBu; branchedC₃₋₄alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclicC₃₋₄alkyl groups, such as -cPr and -cBu.

In some embodiments, C₂₋₄alkenyl groups may be selected from linearC₁₋₄alkenyl groups, such as —CH═CH₂ (vinyl) and —CH₂—CH═CH₂ (allyl).

In some embodiments, halogenated C₁₋₄alkyl groups may be selected from:—CF₃, —CH₂CF₃, and —CF₂CF₃.

The term “C₆₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of aC₆₋₁₀ aromatic compound, said compound having one ring, or two or morerings (e.g., fused), and having from 6 to 10 ring atoms, and wherein atleast one of said ring(s) is an aromatic ring.

The term “C₆₋₁₀ arylene”, as used herein, pertains to a bidentate moietyobtained by removing two hydrogen atoms from an aromatic compound havingfrom 6 to 10 carbon atoms.

In some embodiments, C₆₋₁₀ aryl groups may be selected from C₆₋₁₀carboaryl groups such as phenyl, and naphthyl.

In some embodiments, C₆₋₁₀ arylene groups may be selected from phenyleneand naphthylene.

Said C₁₋₄ alkyl and C₁₋₆ alkylene groups may be unsubstituted or mayoptionally be substituted, for example with one or more groups selectedfrom halo (e.g. F, Cl, Br, or I), amino (e.g. —NH₂, —NHR, or —NR₂,wherein each R is independently C₁₋₄alkyl), hydroxy (—OH), alkoxy (—OR,wherein R is independently C₁₋₄alkyl), nitro (—NO₂), etc.

Said C₆₋₁₀ aryl and C₆₋₁₀ arylene groups may be unsubstituted or mayoptionally be substituted, for example with one or more groups selectedfrom C₁₋₄ alkyl, for example-Me, halogenated C₁₋₄alkyl, for example—CF₃,halo (e.g. F, Cl, Br, or I), amino (e.g. —NH₂, —NHR, or —NR₂, whereineach R is independently C₁₋₄alkyl), hydroxy (—OH), alkoxy (—OR, whereinR is independently C₁₋₄alkyl), nitro (—NO₂), etc.

Groups R^(A) and R^(B)

Each of R^(A) and R^(B) is independently selected from:

C₁₋₄alkyl, halogenated C₁₋₄alkyl, and C₆₋₁₀aryl;

or

R^(A) and R^(B) are linked to form a group R^(AB), wherein R^(AB) isselected from:

C₁₋₆ alkylene and C₆₋₁₀ arylene;

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom:

C₁₋₄alkyl, halogenated C₁₋₄alkyl, and C₆₋₁₀aryl.

In some embodiments, each of R^(A) and R^(B) is independently C₁₋₄alkyl.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom Me, Et, nPr, iPr, nBu, iBu, tBu.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom Me and Et.

In some embodiments, each of R^(A) and R^(B) is independently C₆₋₁₀aryl.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom benzene, 1-naphthalene, 2-naphthalene and p-toluene.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom Me, Et, benzene and p-toluene.

In some embodiments, R^(A) and R^(B) are the same.

In some embodiments, R^(A) and R^(B) are different.

In some embodiments, R^(A) and R^(B) are the same and are independentlyMe. The compound may then be referred to as a diaminophenothiazinebis(methanesulfonate) salt which is of general formula (Ia):

In some embodiments, R^(A) and R^(B) are linked to form a group R^(AB).

In these embodiments, the compounds of the invention may alternately berepresented by general formula Ib:

wherein R^(AB) is selected from C₁₋₆ alkylene and C₆₋₁₀ arylene.

In some embodiments, R^(AB) is a C₁₋₆ alkylene group.

In some embodiments, R^(AB) is a C₁₋₆ alkylene group selected from—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂— and—CH₂CH₂CH₂CH₂CH₂CH₂—.

In some embodiments, R^(AB) is a C₁₋₆ alkylene group selected frommethylene (—CH₂—), ethylene (—CH₂CH₂—) and propylene (—CH₂CH₂CH₂—).

In some embodiments, R^(AB) is ethylene.

In some embodiments, R^(AB) is a C₆₋₁₀ arylene group.

In some embodiments, R^(AB) is a C₆₋₁₀ arylene group selected fromphenylene and naphthylene.

In some embodiments, R^(AB) is phenylene.

In some embodiments, R^(AB) is selected from 1,2-phenylene,1,3-phenylene, and 1,4-phenylene.

In some embodiments, R^(AB) is phenylene optionally substituted with oneor more substituents, for example selected from C₁₋₄ alkyl, halogenatedC₁₋₄alkyl, and halo.

In some embodiments, R^(AB) is naphthylene.

In some embodiments, R^(AB) is selected from 1,2-naphthylene,1,3-naphthylene, 1,4-naphthylene, 1,5-naphthylene, 1,6-naphthylene,1,7-naphthylene and 1,8-naphthylene.

In some embodiments, R^(AB) is selected from:

1,5-naphthylene i.e.

and 1,8-naphthylene. i.e.

In some embodiments, R^(AB) is naphthylene optionally substituted withone or more substituents, for example selected from C₁₋₄ alkyl,halogenated C₁₋₄alkyl, and halo.

Groups R¹ and R⁹

In some embodiments, each of R¹ and R⁹ is independently —H, -Me, -Et, or—CF₃.

In some embodiments, each of R¹ and R⁹ is independently —H, -Me, or -Et.

In some embodiments, R¹ and R⁹ are the same.

In some embodiments, R¹ and R⁹ are different.

In some embodiments, each of R¹ and R⁹ is independently —H.

In some embodiments, each of R¹ and R⁹ is independently -Me.

In some embodiments, each of R¹ and R⁹ is independently -Et.

Groups R^(3NA) and R^(3NB)

Each of R^(3NA) and R^(3NB) is independently selected from: —H,C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In some embodiments, each of R^(3NA) and R^(3NB) is independentlyselected from: C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In some embodiments, each of R^(3NA) and R^(3NB) is independently -Me,-Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In some embodiments, each of R^(3NA) and R^(3NB) is independently -Me,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In some embodiments, each of R^(3NA) and R^(3NB) is independently -Me or-Et.

In some embodiments, R^(3NA) and R^(3NB) are the same.

In some embodiments, R^(3NA) and R^(3NB) are different.

In some embodiments, each of R^(3NA) and R^(3NB) is independently -Me.

Groups R^(7NA) and R^(7NB)

Each of R^(7NA) and R^(7NB) is independently selected from: —H,C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In some embodiments, each of R^(7NA) and R^(7NB) is independentlyselected from: C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In some embodiments, each of R^(7NA) and R^(7NB) is independently -Me,-Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In some embodiments, each of R^(7NA) and R^(7NB) is independently -Me,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In some embodiments, each of R^(7NA) and R^(7NB) is independently -Me or-Et.

In some embodiments, R^(7NA) and R^(7NB) are the same.

In some embodiments, R^(7NA) and R⁷NB are different.

In some embodiments, each of R^(7NA) and R^(7NB) is independently -Me.

Groups R^(3NA), R^(3NB), R^(7NA) and R^(7NB)

In some embodiments:

-   each of R^(3NA) and R^(3NB) is independently C₁₋₄alkyl, C₂₋₄alkenyl,    or halogenated C₁₋₄alkyl;-   each of R^(7NA) and R^(7NB) is independently C₁₋₄alkyl, C₂₋₄alkenyl,    or halogenated C₁₋₄alkyl.

In some embodiments:

-   each of R^(3NA) and R^(3NB) is independently -Me, -Et, -nPr, -nBu,    —CH₂—CH═CH₂, or —CF₃;-   each of R^(7NA) and R^(7NB) is independently -Me, -Et, -nPr, -nBu,    —CH₂—CH═CH₂, or —CF₃.

In some embodiments:

-   each of R^(3NA) and R^(3NB) is independently -Me or -Et;-   each of R^(7NA) and R^(7NB) is independently -Me or -Et.

In some embodiments, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are allthe same.

In some embodiments, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are thesame and are all -Me or all -Et.

In some embodiments, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are thesame and are all -Me.

Salts and Solvates

Although the compounds described herein are themselves salts, they mayalso be provided in the form of a mixed salt (i.e., the compound of theinvention in combination with another salt). Such mixed salts areintended to be encompassed by the term “and pharmaceutically acceptablesalts thereof”. Unless otherwise specified, a reference to a particularcompound also includes salts thereof.

The compounds of the invention may also be provided in the form of asolvate or hydrate. The term “solvate” is used herein in theconventional sense to refer to a complex of solute (e.g., compound, saltof compound) and solvent. If the solvent is water, the solvate may beconveniently referred to as a hydrate, for example, a mono-hydrate, adi-hydrate, a tri-hydrate, etc. Unless otherwise specified, anyreference to a compound also includes solvate and hydrate forms thereof.

Naturally, solvates or hydrates of salts of the compounds are alsoencompassed by the present invention.

Isotopic Variation

In some embodiments, one or more carbon atoms of the compound is ¹¹C,¹³C or ¹⁴C.

In some embodiments, one or more carbon atoms of the compound is ¹¹C.

In some embodiments, one or more carbon atoms of the compound is ¹³C.

In some embodiments, one or more carbon atoms of the compound is ¹⁴C.

In some embodiments, one or more nitrogen atoms of the compound is ¹⁵N.

In some embodiments, one or more or all of the carbon atoms of one ormore or all of the groups R^(3NA), R^(3NB), R^(7NA), R^(7NB), R¹, R⁹,R^(A) and R^(B) is ¹¹C, ¹³C, or ¹⁴C.

In some embodiments, one or more or all of the carbon atoms of one ormore or all of the groups R³NA, R^(3NB), R^(7NA) and R^(7NB) is ¹¹C,¹³C, or ¹⁴C.

Combinations

All compatible combinations of the embodiments described above areexplicitly disclosed herein as if each combination was specifically andindividually recited.

In particular, in the compounds of the invention, the groups R^(3NA),R^(3NB), R^(7NA), R^(7NB), R¹, R⁹, R^(A) and R^(B) (and R^(AB)) aredefined as independent variables and it will be recognised by thoseskilled in the art that any compatible combination of these groups andsubstituents may be utilised in the compounds and methods of the presentinvention.

All compatible combinations of these and other defined variables aretherefore specifically embraced by the present invention, and aredisclosed herein as if each and every combination were individually andexplicitly recited.

Some Preferred Embodiments

In some embodiments, the compound of the invention may be selected fromthe following compounds and pharmaceutically acceptable salts, solvates,and hydrates thereof:

1

LMT•2MsOH (LMTM) 2

LMT•2EsOH 3

LMT•2TsOH 4

LMT•2BSA 5

LMT•EDSA 6

LMT•PDSA 7

LMT•NDSA

One particular compound of the invention is compound 1:

-   N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium    bis(methanesulfonate).

This compound may also be referred to as:

-   N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine    bis(hydromethanesulfonate)-   Leuco methylthioninium bis(hydromethanesulfonate)-   Leuco methylthioninium bis(mesylate)-   LMTM-   LMT.2MsOH    Purity

The compounds of the present invention may conveniently be described asbeing in a “stabilized reduced form”. The compounds oxidize (e.g.,autoxidize) to give the corresponding oxidized forms. Thus, it islikely, if not inevitable, that compositions comprising the compounds ofthe present invention will contain, as an impurity, at least some of thecorresponding oxidized compound.

Thus, another aspect of the present invention pertains to compounds asdescribed herein, in substantially purified form and/or in a formsubstantially free from contaminants (e.g., the corresponding oxidizedcompound, other contaminants).

In some embodiments, the substantially purified form is at least 50% byweight pure, e.g., at least 60% by weight pure, e.g., at least 70% byweight pure, e.g., at least 80% by weight pure, e.g., at least 90% byweight pure, e.g., at least 95% by weight pure, e.g., at least 97% byweight pure, e.g., at least 98% by weight pure, e.g., at least 99% byweight pure.

In some embodiments, the contaminants represent no more than 50% byweight, e.g., no more than 40% by weight, e.g., no more than 30% byweight, e.g., no more than 20% by weight, e.g., no more than 10% byweight, e.g., no more than 5% by weight, e.g., no more than 3% byweight, e.g., no more than 2% by weight, e.g., no more than 1% byweight.

Product-by-Process

In some embodiments, the compound is one which is obtained by, or isobtainable by, a method as described herein.

CHEMICAL SYNTHESIS

Methods for the chemical synthesis of the compounds of the presentinvention are described herein. These and/or other well known methodsmay be modified and/or adapted in known ways in order to facilitate thesynthesis of additional compounds within the scope of the presentinvention.

Compounds of formula (I):

may be prepared from compounds of formula (II):

wherein R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) are as definedpreviously.

Compounds of formula (II) may, for example, be prepared from compoundsof formula (III):

wherein R^(Prot) is an amine protecting group and R¹, R⁹, R^(3NA),R^(3NB), R^(7NA), R^(7NB), R^(A) and R^(B) are as defined previously.

By way of non-limiting example, R^(Prot) may be an acyl group, forexample an acetyl (—C(═O)Me) or a benzoyl (—C(═O)Ph) group.

The compounds of formula (II) may be prepared e.g. by deprotection ofthe compounds of formula (III), or by other known methods. Conversely,compounds of formula (II) may be produced by protection of compounds offormula (III).

Compounds of formulae (II) and (III) are known, and may be prepared fromknown and/or commercially available starting materials, e.g. fromcorresponding phenothiazine compounds, using known methods.

For example, intermediates of formula (II) and (III) were used in themethods for the synthesis of 3,7-diamino-10H-phenothiazinehydrochloride, hydrobromide, and hydroiodide salts disclosed inWO2007/110627.

As disclosed in that document, a suitable phenothiazine can be convertedto the corresponding 3,7-dinitro-phenothiazine, for example using sodiumnitrite with acetic acid and chloroform.

The ring amino group may then be protected, for example as the acetate,for example using acetic anhydride and pyridine.

The nitro groups may then be reduced to amino groups, for example usingtin (II) chloride with ethanol.

The amino groups may then be substituted, for example disubstituted, forexample methyl disubstituted, for example using methyl iodide, sodiumhydroxide, DMSO, and tetra-n-butyl ammonium bromide, to provide aN-acetyl protected 3,7-dialkylamino-10H-phenothiazine.

Examples of such a method are illustrated in Schemes 1a and 1b. The useof any one or more of the reagents described herein in the process is ofcourse encompassed by the present invention:

The amino group of this N-acetyl intermediate can then be deprotected,i.e. the N-acetyl group may be removed, for example using aqueous acid.

Compounds of formulae (II) and (III) may also be made using the methodsdisclosed in WO2008/007074. This document discloses compounds of formula(III) and compounds of formula (II) wherein R^(Prot) is an acyl group,for example an acetyl group.

In one approach, an appropriate thioninium chloride (e.g., methylthioninium chloride, ethyl thioninium chloride, etc) may first bereduced and acetylated to give the corresponding1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone, for example, byreaction with hydrazine (NH₂NH₂), methyl hydrazine (MeNHNH₂), or sodiumborohydride (NaBH₄); and acetic anhydride ((H₃CCO)₂O); for example, inthe presence of a suitable base, for example, pyridine (C₅H₅N) orHünig's base (diisopropylethylamine, C₈H₁₉N), for example, in a suitablesolvent, for example, ethanol or acetonitrile. The reduced andacetylated compound (of formula (III)) may then be deprotected (byremoving the acetyl group), for example by reaction with a suitableacid, to give a compound of formula (II) or may be used directly.Advantageously, this reaction may produce a product with a high degreeof purity.

An example is shown in the following scheme.

In another approach, an appropriate thioninium salt, for example, ethylthioninium semi zinc chloride, may be simultaneously reduced and thering amino group protected, for example, by reaction with a reducingagent phenylhydrazine, ethanol, acetic anhydride, and pyridine.

An example is shown in the following scheme:

In one aspect, the present invention therefore provides a method ofpreparing a 3,7-diamino-10H-phenothiazine compound of formula (I):

from a compound of formula (II):

wherein R^(A), R^(B), R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) areas previously defined.

In some embodiments, the method comprises the step of:

-   -   salt formation (SF).

In some embodiments, salt formation (SF) comprises treatment of acompound of formula (II) with an appropriate sulfonic acid.

In some embodiments, salt formation comprises treatment of a solution ofa compound of formula (II) with an appropriate sulfonic acid, in anorganic solvent.

In a further aspect, the present invention provides a method ofpreparing a 3,7-diamino-10H-phenothiazine compound of formula (I):

from a compound of formula (III):

wherein R^(A), R^(B), R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) areas previously defined and wherein R^(Prot) is an amine protecting group.

A wide variety of amine protecting groups are widely used and well knownin organic synthesis. See, for example, Protective Groups in OrganicSynthesis (T. Green and P. Wuts; 4th Edition; John Wiley and Sons,2006).

In some embodiments, the amine protecting group is an acid-cleavableprotecting group.

In some embodiments, the amine protecting group is an acyl group, suchas an acetyl group.

In some embodiments, the method comprises the steps of:

-   -   ring amino deprotection (DP); and    -   salt formation (SF).

Ring amino deprotection (DP) comprises removal of the protecting groupto convert the N-protected ring amine group (—NR^(Prot)—) to a free ringamine group (—NH—). Deprotection of a compound of formula (III) producesthe corresponding compound of formula (II).

Methods for the removal of amine protecting groups are known in the art.See, for example, Protective Groups in Organic Synthesis (T. Green andP. Wuts; 4th Edition; John Wiley and Sons, 2006).

In some embodiments, the step of ring amino deprotection (DP) and thestep of salt formation (SF) are performed simultaneously (i.e., as onestep). For example:

In some embodiments, simultaneous ring amino deprotection (DP) and saltformation (SF) comprises treatment of the compound of formula (III) withan appropriate sulfonic acid, to produce a bis(sulfonate) salt offormula (I).

In some embodiments, simultaneous ring amine deprotection and saltformation may comprise treatment of a solution of a compound of formula(III) in an organic solvent with the sulfonic acid and water.

In some embodiments, the organic solvent is toluene.

In the methods of the invention, the sulfonic acid may be selected fromalkylsulfonic acids and arylsulfonic acids. It may be a sulfonic acid offormula R^(A)SO₃H or R^(B)SO₃H, wherein R^(A) and R^(B) are as definedherein.

In some embodiments, the sulfonic acid may be a disulfonic acid. i.e. acompound containing two sulfonic acid moieties per molecule. Thesesulfonic acid moieties may be linked by e.g. an alkylene or arylenegroup.

In some embodiments the sulfonic acid may be selected from:methanesulfonic acid (MsOH), ethanesulfonic acid (EsOH), benzenesulfonicacid (BSA), naphthalenesulfonic acid (NSA), p-toluenesulfonic acid(TsOH), ethanedisulfonic acid (EDSA), propanedisulfonic acid (PDSA) andnaphthalene-1,5-disulfonic acid (NDSA).

In some embodiments, the phenothiazine starting material (i.e. thecompound of formula (III) is first heated in said organic solvent untilcompletely dissolved and the resultant solution is filtered beforeaddition of the reagents (i.e. the sulfonic acid and water).

In some embodiments, the compound is heated in said organic solvent at atemperature of about 60-80° C., for example at a temperature of about70° C.

In some embodiments, the sulfonic acid is added in an amount of at least2 molar equivalents, for example about 2.2 molar equivalents, relativeto the phenothiazine starting material. If a disulfonic acid is used, itwill be understood the molar amount of the acid will be at least 1 molarequivalent, for example about 1.1 molar equivalents, so as to achievethe same number of sulfonic acid moieties per molecule of phenothiazinestarting material.

It may be desirable to add the sulfonic acid slowly to prevent atemperature increase (exotherm). Therefore, in some embodiments, thesulfonic acid is added gradually.

In some embodiments, the sulfonic acid is added at a temperature ofabout 15-25° C.

In some embodiments, after addition of the sulfonic acid and water, thereaction is heated to a temperature of about 80-90° C.

In some embodiments, the reaction is maintained at this temperatureuntil judged complete by e.g. chromatographic analysis.

In some embodiments, after reaction, the solution is treated with acounter solvent to precipitate the product. In some embodiments, thecounter solvent is an alcohol, for example ethanol.

It may be desirable to ‘seed’ the reaction mixture with a small amount,for example, about 1 mg per gram of starting material (compound offormula (II), of the desired bis(sulfonate) product. Without wishing tobe bound by theory, it is thought that addition of the seed ensuresearly and effificent precipitation of the desired product, reducing theopportunity for possible side-reactions and by-product formation. Theseed is also thought to be of use in controlling the particle size ofthe precipitated product.

Hence in some embodiments, after reaction, the resultant mixture isseeded with a small amount of the desired bis(sulfonate) salt.

In some embodiments, the seed comprises particles of the desiredbis(sulfonate) salt which have been ground.

In some embodiments, the seed comprises particles of the desiredbis(sulfonate) salt which have been ground to a size of less than about100 μm.

In some embodiments, the precipitated product is isolated by filtration.

In some embodiments, after filtration, the product is washed with anorganic solvent, for example ethanol or acetonitrile. Salt formation(SF) produces the bis(sulfonate) salt of formula (I) from the compoundof formula (II):

As explained above, the bis(sulfonate) salt may also be prepareddirectly from a corresponding amino-protected (e.g. N-acetyl) compoundof formula (III).

In this case, salt formation may be performed at the same time asdeprotection, for example by using the appropriate sulfonic acid, e.g.methanesulfonic acid, for the deprotection step. An example isillustrated in the following scheme:

In a further aspect, the present invention provides a method ofpreparing a compound of formula (I):

wherein R^(A), R^(B), R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) areas previously defined the method comprising:

-   -   preparing a compound of formula (II) or (III) as defined herein,        followed by    -   salt formation (SF) and/or    -   ring amine deprotection (DP).

The steps of salt formation (SF) and ring amine deprotection (DP) are asdescribed above.

In some embodiments, preparing said compound of formula (II) or (III)comprises a method as disclosed in WO2007/110627.

In some embodiments, preparing said compound of formula (II) or (III)comprises a method as disclosed in WO2008/007074.

In some embodiments, preparing a compound of formula (II) comprises ringamine deprotection (DP) of a compound of formula (III), as set outabove.

In some embodiments, preparing a compound of formula (III) comprises oneor more steps selected from:

-   -   nitration (NO),    -   ring amino protection (AP),    -   nitro reduction (NR),    -   amine substitution (AS).

In some embodiments, preparing a compound of formula (III) comprises thesteps of

-   -   reduction (RED), and    -   ring amino protection (AP).

The steps may be performed in any logical order. In some embodiments,the steps are performed in the order listed (i.e., any step in the listis performed at the same time as, or subsequent to, the preceding stepin the list).

In some embodiments, nitration (NO) comprises:

-   -   nitration (NO), wherein a 10H-phenothiazine is converted to a        3,7-dinitro-10H-phenothiazine, for example:

In some embodiments, nitration is performed using a nitrite, forexample, sodium nitrite, for example, sodium nitrite with acetic acid,and a solvent such as dimethyl sulfoxide, dimethyl formamide,acetonitirle, tetrahydrofuran, dimethoxyethane, acetone, dichloromethaneor chloroform.

In some embodiments, ring amino protection (AP) comprises:

-   -   ring amino protection (AP), wherein the ring amino group (—NH—)        of a 3,7-dinitro-10H-phenothiazine is converted to a protected        ring amino group (—NR^(prot)), for example:

In some embodiments, ring amino protection is achieved as an acetate,for example, using acetic anhydride, for example, using acetic anhydrideand a base such as an amine base, for example triethylamine or pyridine.

In some embodiments, nitro reduction (NR) step comprises:

-   -   nitro reduction (NR), wherein each of the nitro (—NO₂) groups of        a protected 3,7-dinitro-10H-phenothiazine is converted to an        amino (—NH₂) group, for example:

In some embodiments, nitro reduction may be performed using, forexample, tin (II) chloride, for example, tin (II) chloride with ethanol.

In some embodiments, nitro reduction may be performed using, forexample, palladium on charcoal (Pd/C) and hydrogen in, for example,2-methyl-tetrahydrofuran.

In some embodiments, nitro reduction may be performed using, forexample, zinc and aqueous ammonium chloride in methanol and THF

In some embodiments, amine substitution (AS) step comprises:

-   -   amine substitution (AS), wherein each of the amino (—NH₂) groups        of a protected 3,7-diamino-10H-phenothiazine is converted to        disubstituted amino group, for example:

In some embodiments, amine substitution is performed using an alkylhalide, for example, an alkyl iodide, for example, methyl iodide, forexample, methyl iodide with sodium hydroxide, DMSO, toluene andtetra-n-butyl ammonium bromide.

In some embodiments, amine substitution comprises treatment withformaldehyde (e.g. paraformaldehyde, formalin) under reducingconditions. For example, treatment with formalin and hydrogen gas, inthe presence of a Pd/C catalyst; or treatment with paraformaldehyde inthe presence of a reducing agent such as sodium cyanoborohydride andacetic acid.

In some embodiments, the reduction (RED) step is:

-   -   reduction (RED), wherein a 3,7-di(disubstituted        amino)-thioninium salt is reduced to give the corresponding        3,7-di(disubstituted amino)-10H-phenothiazine, for example by        treatment with a reducing agent, such as hydrazine (NH₂NH₂),        methyl hydrazine (MeNHNH₂), or sodium borohydride and a base,        such as pyridine, triethylamine, or Hünig's base        (diisopropylethylamine).

In some embodiments, the ring amino protection (AP) step is:

-   -   ring amino protection (AP), wherein a 3,7-di(disubstituted        amino)-10H-phenothiazine is protected, for example by treatment        with acetic anhydride, to give the corresponding protected        3,7-di(disubstituted amino)-10H-phenothiazine, for example the        corresponding N-acetyl 3,7-di(disubstituted        amino)-10H-phenothiazine.

In some embodiments, the steps are performed in the order listed (i.e.,any step in the list is performed at the same time as, or subsequent to,the preceding step in the list).

In some embodiments, the step of reduction (RED) and the step of ringamino protection (AP) are performed simultaneously (i.e., as one step).

For example, in some embodiments, the combined reduction (RED) step andring amino protection (AP) step is:

-   -   reduction (RED) and ring amino protection (AP), wherein a        3,7-di(disubstituted amino)-thioninium salt is reduced to give        the corresponding 3,7-di(disubstituted amino)-10H-phenothiazine,        and the ring amino group (—NH—) of the 3,7-di(disubstituted        amino)-10H-phenothiazine is converted to a protected ring amino        group (—R^(Prot)) to give the corresponding protected        3,7-di(disubstituted amino)-10H-phenothiazine, for example:

wherein Y is a counterion. In some embodiments, Y represents Cl′.

In some embodiments, the 3,7-di(disubstituted amino)-thioninium salt ismethylthioninium chloride (MTC).

In some embodiments, the combined reduction (RED) step and ring aminoprotection (AP) step is achieved using a hydrazine, such asphenylhydrazine, MeNHNH₂, or NH₂NH₂.H₂O and acetic anhydride.

In some embodiments, the step is performed under a nitrogen atmosphere.

In some embodiments the combined reduction (RED) step and ring aminoprotection (AP) step is performed using, for example, phenylhydrazine,ethanol, acetic anhydride, and pyridine.

In some embodiments, the combined reduction (RED) step and ring aminoprotection (AP) step is performed using, for example, hydrazine hydrate,acetonitrile, acetic anhydride, and triethylamine, under a nitrogenatmosphere.

In some embodiments, the protected 3,7-di(disubstitutedamino)-10H-phenothiazine, for example the N-acetyl 3,7-di(disubstitutedamino)-10H-phenothiazine, undergoes a purification step.

In some embodiments, purification comprises addition of an organicsolvent, for example toluene, and an acid, for example acetic acid, todissolve the compound, followed by a washing step.

In some embodiments, washing comprises addition of water and/or aqueousacetic acid to the solution of the compound; agitation and/or heating;and separation of the organic layer.

In some embodiments, washing is repeated, for example up to three times.

In some embodiments, washing is followed by isolation of the purifiedproduct.

In some embodiments, isolation of the purified product comprisescooling, precipitation and filtration of the product.

Crystalline Forms

In some embodiments, the compound of the invention is provided incrystalline form.

In some embodiments, the crystalline form is ‘Form A’ as describedherein.

In some embodiments, the crystalline form has the structure depicted inFIG. 17 and\or is characterised by the crystal data shown in an AnnexTable 1 and\or the atomic co-ordinates shown in an Annex Table 2 and\orthe bond lengths and angles shown in an Annex Table 3 and\or theanisotropic displacement parameters shown in an Annex Table 4 and\or thehydrogen coordinates and isotropic displacement parameters shown in anAnnex Table 5.

Reversing and/or Inhibiting the Aggregation of a Protein

One aspect of the invention is the use of a compound or composition asdescribed herein, to regulate (e.g., to reverse and/or inhibit) theaggregation of a protein, for example, aggregation of a proteinassociated with a neurodegenerative disease and/or clinical dementia.The aggregation may be in vitro, or in vivo, and may be associated witha disease state as discussed below.

Thus, one aspect of the invention pertains to a method of regulating(e.g., reversing and/or inhibiting) the aggregation of a protein, forexample, aggregation of a protein associated with a neurodegenerativedisease and/or clinical dementia, comprising contacting the protein withan effective amount of a compound or composition as described herein.The method may be performed in vitro, or in vivo.

Similarly, one aspect of the invention pertains to a method ofregulating (e.g., reversing and/or inhibiting) the aggregation of aprotein in the brain of a mammal, which aggregation is associated with adisease state as described herein, the treatment comprising the step ofadministering to said mammal in need of said treatment, aprophylactically or therapeutically effective amount of a compound orcomposition as described herein, that is an inhibitor of saidaggregation.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment comprising administering to a patient in need of treatment aprophylactically or therapeutically effective amount of a compound asdescribed herein, preferably in the form of a pharmaceuticalcomposition.

Use in Methods of Therapy

Another aspect of the present invention pertains to a compound orcomposition as described herein, for use in a method of treatment (e.g.,of a disease condition) of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a compound orcomposition as described herein, in the manufacture of a medicament foruse in treatment (e.g., of a disease condition).

In some embodiments, the medicament comprises a compound of theinvention.

In some embodiments, the medicament is a composition as describedhereinbelow.

Disease Conditions Treated—Diseases of Protein Aggregation

The compounds and compositions of the present invention are useful inthe treatment or prophylaxis of diseases of protein aggregation.

Thus, in some embodiments, the disease condition is a disease of proteinaggregation, and, for example, the treatment is with an amount of acompound or composition as described herein, sufficient to inhibit theaggregation of the protein associated with said disease condition.

In general, the protein aggregation is that which arises from an inducedconformational polymerisation interaction, i.e., one in which aconformational change of the protein, or in a fragment thereof, givesrise to templated binding and aggregation of further (precursor) proteinmolecules in a self-propagating manner. Once nucleation is initiated, anaggregation cascade may ensue which involves the induced conformationalpolymerisation of further protein molecules, leading to the formation oftoxic product fragments in aggregates which are substantially resistantto further proteolysis. The protein aggregates thus formed are thoughtto be a proximal cause of disease states manifested asneurodegeneration, clinical dementia, and other pathological symptoms.

The following Table lists various disease-associated aggregatingproteins and the corresponding diseases of protein aggregation. The useof the compounds and compositions of the invention in respect of theseproteins or diseases is encompassed by the present invention.

Diseases of protein aggregation Fibril Aggregating subunit domain and/orsize Protein Disease mutations (kDa) Reference Neurodegenerativedisorders Prion protein Prion diseases Inherited and 27 Prusiner (1998)sporadic forms (CJD, nvCJD, Fatal PrP-27-30; many 27 Prusiner (1998)familial insomnia, mutations. Gerstmann-Straussler- Scheinker syndrome,Kuru) Fibrillogenic Gasset et al. domains: 113-120, (1992) 178-191,202-218. Tau protein Alzheimer's disease, Inherited and 10-12 Wischik etal. Down's syndrome, FTDP- sporadic forms (1988) 17, CBD,post-encephalitic parkinsonism, Pick's disease, parkinsonism withdementia complex of Guam Truncated tau 10-12 Wischik et al.(tubulin-binding (1988) domain) 297-391. Mutations in tau Hutton et al.(1998) in FTDP-17. Many mutations Czech et al. (2000) in presenilinproteins. Amyloid Alzheimer's disease, Inherited and 4 Glenner & Wong,β-protein Down's syndrome sporadic forms (1984) Amyloid β- 4 Glenner &Wong, protein; 1-42(3). (1984) Mutations in APP Goate et al. (1991) inrare families. Huntingtin Huntington's disease N-termini of 40 DiFigliaet al. protein with (1997) expanded glutamine repeats. AtaxinsSpinocerebellar ataxias Proteins with Paulson et al. (1, 2, 3, 7) (SCA1,2, 3, 7) expanded (1999) glutamine repeats. AtrophinDentatorubropallidoluysian Proteins with Paulson et al. atrophy (DRPLA)expanded (1999) glutamine repeats. Androgen Spinal and bulbar Proteinswith Paulson et al. receptor muscular atrophy expanded (1999) glutaminerepeats. Neuroserpin Familial encephalopathy Neuroserpin; 57 Davis etal. (1999) with neuronal inclusion S49P, S52R. bodies (FENIB)α-Synuclein Parkinson's disease, Inherited and 19 Spillantini et al.dementia with Lewy sporadic forms (1998) also bodies, multiple systemPCT/GB2007/001105 atrophy A53T, A30P in Polymeropoulos et rareautosomal- al. (1997) dominant PD families. TDP-43 FTLD-TDP SeveralTDP-43 10-43 Mackenzie et al. mutations (2010) Amyotrophic lateralSeveral TDP-43 10-43 Mackenzie et al. sclerosis mutations (2010)Cystatin C Hereditary cerebral Cystatin C less 12-13 Abrahamson et al.angiopathy (Icelandic) 10 residues; (1992) L68Q. Superoxide Amyotrophiclateral SOD1 mutations. 16 Shibata et al. dismutase 1 sclerosis (1996)Non-neurodegenerative disorders Haemoglobin Sickle cell anaemiaHaemoglobin Carrell & Gooptu beta chain (S). (1998) Inclusion bodyhaemolysis Many mutations. Serpins α1-Antitrypsin deficiency MutationsLomas et al. (1992) (emphysema, cirrhosis) Antithrombin deficiencyMutatons Carrell & Gooptu (thromboembolic disease) (1998) C1-inhibitordeficiency Mutations Carrell & Gooptu (angioedema) (1998) ImmunoglobulinPlasma cell dyscrasias Light chain or 0.5-25  Westermark et al. lightchain (primary systemic AL fragments. (1985) amyloidosis) Serum amyloidA Reactive, secondary 76-residue 4.5-7.5 Westermark et al. systemic AAamyloidosis fragment (critical (1985) residues 2-12). Chronicinflammatory disease Transthyretin Familial amyloid Tetramer 10-14Gustavsson et al. polyneuropathy (systemic; dissociated to (1991) FAP I)conformational monomer variant. Many mutations (some not associated withamyloid; several different types of disease). Senile cardiac amyloidosisNormal 10-14 Gustavsson et al. transthyretin (1991) Gelsolin Familialamyloidosis - D187Q leads to 9.5 Maury & Baumann Finnish type (FAP IV)truncated 173-225/ (1990) 243 (critical residues 182-192). β2-Haemodialysis β2-Microglobulin 12-25 Gorevic et al. Microglobulinamyloidosis (1985) Prostatic amyloid Apolipoprotein Familial amyloidN-terminal 83-93 9 Booth et al. (1997) AI polyneuropathy (systemic;residues; G26R, FAP III) W50R, L60R Lysozyme Familial visceral Lysozymeor 14 Pepys et al. (1993) amyloidosis fragments (with or without I56T,D67H) Amylin (Islet Type II diabetes (NIDDM) Fragments 3.9 Westermark(1990) amyloid (critical core of polypeptide) 20-29); no mutationsFibrinogen Hereditary renal Fibrinogen  7-10 Uemichi et al. α-chainamyloidosis fragments (1992) Procalcitonin Medullary carcinoma ofCalcitonin 3.4 Sletten et al. thyroid fragments (1976) Atrialnatriuretic Cardiac amyloidosis ANF, no mutants 3.5 Johansson et al.factor (1987) Insulin Injection localised Insulin Dische et al. (1988)amyloidosis Multiple Inclusion body myositis β-amyloid, tau, Askenas etat proteins ubiquitin, ApoE, (2009) and presenilin-1 Other proteins (invitro) Other proteins Chiti et al. (1999) forming amyloid As describedin WO 02/055720, WO2007/110630, and WO2007/110627, diaminophenothiazineshave utility in the inhibition of such protein aggregating diseases.

Thus it will be appreciated that, except where context requiresotherwise, description of embodiments with respect to tau protein ortau-like proteins (e.g., MAP2; see below), should be taken as applyingequally to the other proteins discussed herein (e.g., β-amyloid,synuclein, prion, etc.) or other proteins which may initiate or undergoa similar pathological aggregation by virtue of conformational change ina domain critical for propagation of the aggregation, or which impartsproteolytic stability to the aggregate thus formed (see, e.g., thearticle by Wischik et al. in “Neurobiology of Alzheimer's Disease”, 2ndEdition, 2000, Eds. Dawbarn, D. and Allen, S. J., The Molecular andCellular Neurobiology Series, Bios Scientific Publishers, Oxford). Allsuch proteins may be referred to herein as “aggregating diseaseproteins.”

Likewise, where mention is made herein of “tau-tau aggregation”, or thelike, this may also be taken to be applicable to other“aggregating-protein aggregation”, such as β-amyloid aggregation, prionaggregation, synuclein aggregation, etc. The same applies for “tauproteolytic degradation” etc.

Preferred Aggregating Disease Proteins

Preferred embodiments of the invention are based on tau protein. Theterm “tau protein,” as used herein, refers generally to any protein ofthe tau protein family. Tau proteins are characterised as being oneamong a larger number of protein families which co-purify withmicrotubules during repeated cycles of assembly and disassembly (see,e.g., Shelanski et al., 1973, Proc. Natl. Acad. Sci. USA, Vol. 70, pp.765-768), and are known as microtubule-associated-proteins (MAPs).Members of the tau family share the common features of having acharacteristic N-terminal segment, sequences of approximately 50 aminoacids inserted in the N-terminal segment, which are developmentallyregulated in the brain, a characteristic tandem repeat region consistingof 3 or 4 tandem repeats of 31-32 amino acids, and a C-terminal tail.

MAP2 is the predominant microtubule-associated protein in thesomatodendritic compartment (see, e.g., Matus, A., in “Microtubules”[Hyams and Lloyd, Eds.] pp. 155-166, John Wiley and Sons, New York,USA). MAP2 isoforms are almost identical to tau protein in the tandemrepeat region, but differ substantially both in the sequence and extentof the N-terminal domain (see, e.g., Kindler and Garner, 1994, Mol.Brain. Res., Vol. 26, pp. 218-224). Nevertheless, aggregation in thetandem-repeat region is not selective for the tau repeat domain. Thus itwill be appreciated that any discussion herein in relation to tauprotein or tau-tau aggregation should be taken as relating also totau-MAP2 aggregation, MAP2-MAP2 aggregation, and so on.

In some embodiments, the protein is tau protein.

In some embodiments, the protein is a synuclein, e.g., α- orβ-synuclein.

In some embodiments, the protein is TDP-43.

TAR DNA-Binding Protein 43 (TDP-43) is a 414 amino acid protein encodedby TARDBP on chromosome 1p36.2. The protein is highly conserved, widelyexpressed, and predominantly localised to the nucleus but can shuttlebetween the nucleus and cytoplasm (Mackenzie et al 2010). It is involvedin transcription and splicing regulation and may have roles in otherprocesses, such as: microRNA processing, apoptosis, cell division,stabilisation of messenger RNA, regulation of neuronal plasticity andmaintenance of dendritic integrity. Furthermore, since 2006 asubstantial body of evidence has accumulated in support of the TDP-43toxic gain of function hypothesis in amyotrophic lateral sclerosis(ALS). TDP-43 is an inherently aggregation-prone protein and aggregatesformed in vitro are ultrastructurally similar to the TDP-43 depositsseen in degenerating neurones in ALS patients (Johnson et al 2009).Johnson et al (2008) showed that when TDP-43 is overexpressed in a yeastmodel only the aggregated form is toxic. Several in vitro studies havealso shown that C-terminal fragments of TDP-43 are more likely thanfull-length TDP-43 to form insoluble cytoplasmic aggregates that becomeubiquitinated, and toxic to cells (Arai et al 2010; Igaz et al 2009;Nonaka et al 2009; Zhang et al 2009). Though Nonaka et al (2009)suggested that these cytoplasmic aggregates bind the endogenousfull-length protein depleting it from the nucleus, Zhang et al (2009)found retention of normal nuclear expression, suggesting a purely toxiceffect for the aggregates. Yang et al (2010) have described the captureof full-length TDP-43 within aggregates of C- and N-terminal fragmentsof TDP-43 in NSC34 motor neurons in culture. Neurite outgrowth, impairedas a result of the presence of such truncated fragments, could berescued by overexpression of the full-length protein. Although the roleof neurite outgrowth in vivo has not been established, this model wouldsupport the suggestion made by Nonaka and colleagues for a role ofTDP-43 aggregation in ALS pathogenesis.

Mutant TDP-43 expression in cell cultures has repeatedly been reportedto result in increased generation of C-terminal fragments, with evengreater cytoplasmic aggregation and toxic effects than the wild-typeprotein (Kabashi et al 2008; Sreedharan et al 2008; Johnson et al 2009;Nonaka et al 2009; Arai et al 2010; Barmarda et al 2010; Kabashi et al2010).

Where the protein is tau protein, in some embodiments of the presentinvention, there is provided a method of inhibiting production ofprotein aggregates (e.g. in the form of paired helical filaments (PHFs),optionally in neurofibrillary tangles (NFTs) in the brain of a mammal,the treatment being as described above.

Preferred Indications—Diseases of Protein Aggregation

Notably it is not only Alzheimer's disease (AD) in which tau protein(and aberrant function or processing thereof) may play a role. Thepathogenesis of neurodegenerative disorders such as Pick's disease andprogressive supranuclear palsy (PSP) appears to correlate with anaccumulation of pathological truncated tau aggregates in the dentategyrus and stellate pyramidal cells of the neocortex, respectively. Otherdementias include fronto-temporal dementia (FTD); FTD with parkinsonismlinked to chromosome 17 (FTDP-17);disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration(CBD) and others (see, e.g., the article by Wischik et al. in“Neurobiology of Alzheimer's Disease”, 2nd Edition, 2000, Eds. Dawbarn,D. and Allen, S. J., The Molecular and Cellular Neurobiology Series,Bios Scientific Publishers, Oxford; especially Table 5.1). All of thesediseases, which are characterized primarily or partially by abnormal tauaggregation, are referred to herein as “tauopathies”.

Thus, in some embodiments, the disease condition is a tauopathy.

In some embodiments, the disease condition is a neurodegenerativetauopathy.

In some embodiments, the disease condition is selected from Alzheimer'sdisease (AD), Pick's disease, progressive supranuclear palsy (PSP),fronto temporal dementia (FTD), FTD with parkinsonism linked tochromosome 17 (FTDP 17), frontotemporal lobar degeneration (FTLD)syndromes; disinhibition-dementia-parkinsonism-amyotrophy complex(DDPAC), pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome,pallido nigro luysian degeneration (PNLD), cortico-basal degeneration(CBD), dementia with argyrophilic grains (AgD), dementia pugilistica(DP) or chronic traumatic encephalopathy (CTE), Down's syndrome (DS),dementia with Lewy bodies (DLB), subacute sclerosing panencephalitis(SSPE), MCI, Niemann-Pick disease, type C (NPC), Sanfilippo syndrometype B (mucopolysaccharidosis III B), or myotonic dystrophies (DM), DM1or DM2, or chronic traumatic encephalopathy (CTE).

In some embodiments, the disease condition is a lysosomal storagedisorder with tau pathology. NPC is caused by mutations in the geneNPC1, which affects cholesterol metabolism (Love et al 1995) andSanfilippo syndrome type B is caused by a mutation in the gene NAGLU, inwhich there is lysosomal accumulation of heparin sulphate (Ohmi et al.2009). In these lysosomal storage disorders, tau pathology is observedand its treatment may decrease the progression of the disease. Otherlysosomal storage disorders may also be characterised by accumulation oftau.

Use of phenothiazine diaminium salts in the treatment of Parkinson'sDisease and MCI is described in more detail in PCT/GB2007/001105 andPCT/GB2008/002066.

In some embodiments, the disease condition is Parkinson's Disease, MCI,or Alzheimer's disease.

In some embodiments, the disease condition is Huntington's Disease orother polyglutamine disorder such as spinal bulbar muscular atrophy (orKennedy disease), and dentatorubropallidoluysian atrophy and variousspinocerebellar ataxias.

In some embodiments, the disease condition is an FTLD syndrome (whichmay for example be a tauopathy or TDP-43 proteinopathy, see below).

In some embodiments, the disease condition is PSP or ALS.

In some embodiments, treatment (e.g., treatment of a neurodegenerativetauopathy, e.g., Alzheimer's disease) may optionally be in combinationwith one or more other agents, for example, one or more cholinesteraseinhibitors (such as Donepezil (also known as Aricept™), Rivastigmine(also known as Exelon™), Galantamine (also known as Reminyl™), NMDAreceptor antagonists (such as Memantine (also known as Ebixa™,Namenda™), muscarinic receptor agonists, and/or inhibitors of amyloidprecursor protein processing that leads to enhanced generation ofbeta-amyloid.

TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS;ALS-TDP) and frontotemporal lobar degeneration (FTLD-TDP).

The role of TDP-43 in neurodegeneration in ALS and otherneurodegenerative disorders has been reviewed in several recentpublications (Chen-Plotkin et al 2010; Gendron et al 2010; Geser et al2010; Mackenzie et al 2010).

ALS is a neurodegenerative disease, characterised by progressiveparalysis and muscle wasting, consequent on the degeneration of bothupper and lower motor neurones in the primary motor cortex, brainstemand spinal cord. It is sometimes referred to as motor neuron disease(MND) but there are diseases other than ALS which affect either eitherupper or lower motor neurons. A definite diagnosis requires both upperand lower motor neurone signs in the bulbar, arm and leg musculaturewith clear evidence of clinical progression that can not be explained byany other disease process (Wijesekera and Leigh 2009).

Although the majority of cases are ALS-TDP, there are other cases wherethe pathological protein differs from TDP-43. Misfolded SOD1 is thepathological protein in ubiquitin-positive inclusions in ALS with SOD1mutations (Seetharaman et al 2009) and in a very small subset(approximately 3-4%) of familial ALS, due to mutations in FUS (fused insarcoma protein), the ubiquitinated pathological protein is FUS (Vanceet al 2009; Blair et al 2010). FUS, like TDP-43, appears to be importantin nuclear-cytoplasmic shuttling although the ways in which impairednuclear import of FUS remains unclear. A new molecular classification ofALS, adapted from Mackenzie et al (2010), reflects the distinctunderlying pathological mechanisms in the different subtypes (see Tablebelow).

New Molecular Classification of ALS (modified from Mackenzie et al2010). In the majority of cases, TDP-43 is the pathologicalubiquitinated protein found in ALS.

Ubiquitin-positive inclusions in ALS Ubiquitinated disease proteinTDP-43 FUS SOD1 Clinico- ALS-TDP ALS-FUS ALS-SOD1 pathologic subtypeAssociated TARDBP FUS SOD1 genotype Frequency of ALS Common Rare Rarecases

Amyotrophic lateral sclerosis has been recognised as a nosologicalentity for almost a century and a half and it is recognised in ICD-10 isclassified as a subtype of MND in ICD 10 (G12.2). Reliable clinicaldiagnostic are available for ALS, which differ little from Charcot'soriginal description, and neuropathological criteria, reflecting theunderlying molecular pathology, have also been agreed.

While ALS is classified pathologically into three subgroups, ALS-TDP,ALS-SOD1 and ALS-FUS, both latter conditions are rare. The largest studyto date showed all sporadic ALS cases to have TDP-43 pathology(Mackenzie et al 2007). Only around 5% of ALS is familial (Byrne et al2010) and mutations in SOD1, the commonest mutations found in FALS,account for between 12-23% of cases (Andersen et al 2006). SOD1 may alsobe implicated in 2-7% of SALS. Mutations in FUS appear to be far lesscommon, accounting for only around 3-4% of FALS (Blair et al 2010). Soit can be reliably predicted that a clinical case of SALS will haveTDP-43 based pathology. Similarly this can be reliably predicted in FALSdue to mutations in TDP-43, which account for around 4% of cases(Mackenzie et al 2010). ALS with mutations in: VCP, accounting for 1-2%of FALS (Johnson et al 2010), ANG (Seilhean et al 2009), and CHMP2B (Coxet al 2010) have also been reported to be associated with TDP-43positive pathology. Although SOD1, FUS and ATXN2 mutations have not beenfound to be associated with TDP-43 positive aggregates, it has howeverbeen reported that TDP-43 is implicated in the pathological processesputatively arising from these mutations (Higashi et al 2010; Ling et al2010; Elden et al 2010).

It is therefore established that TDP-43 has an important, andpotentially central role, in the pathogenesis of the vast majority ofSALS cases and may be implicated in the pathogenesis of a significantproportion of FALS. ALS is now widely considered to be a TDP-43proteinopathy (Neumann et al 2009) and numerous in vitro, and in vivostudies provide support to the hypothesis that toxic gain of function,due to TDP-43 aggregation is responsible for at least some of theneurotoxicity in the disease.

FTLD syndromes are insidious onset, inexorably progressive,neurodegenerative conditions, with peak onset in late middle age. Thereis often a positive family history of similar disorders in a firstdegree relative.

Behavioural variant FTD is characterised by early prominent change insocial and interpersonal function, often accompanied by repetitivebehaviours and changes in eating pattern. In semantic dementia there areprominent word finding problems, despite otherwise fluent speech, withdegraded object knowledge and impaired single word comprehension oncognitive assessment. Progressive non-fluent aphasia presents with acombination of motor speech problems and grammatical deficits. The coreclinical diagnostic features for these three FTLD syndromes are shown inthe Table below and the full criteria in Neary et al (1998).

Clinical Profile and Core Diagnostic Features of FTLD Syndromes

FTLD Syndrome-Clinical Profile Core Diagnostic Features FrontotemporalDementia 1. Insidious onset and gradual Character change and disorderedsocial progression conduct are the dominant features initially and 2.Early decline in social interpersonal throughout the disease course.Instrumental conduct functions of perception, spatial skills, praxis 3.Early impairment in regulation of and memory are intact or relativelywell personal conduct preserved. 4. Early emotional blunting 5. Earlyloss of insight Semantic Dementia A) Insidious onset and gradualprogression Semantic disorder (impaired understanding of B) Languagedisorder characterised by word meaning and/or object identity) is the 1.Progressive, fluent empty speech dominant feature initially andthroughout the 2. Loss of word meaning manifest by disease course. Otheraspects of cognition, impaired naming and comprehension includingautobiographic memory, are intact or 3. Semantic paraphasias and/orrelatively well preserved. 4. Perceptual disorder characterised by 1.Prosopagnosia: impaired recognition of identity of familiar faces and/or2. Associative agnosia: impaired recognition of object identity C)Preserved perceptual matching and drawing reproduction D) Preservedsingle word repetition E) Preserved ability to read aloud and write todictation orthographically regular words Progressive Non-fluent AphasiaA) Insidious onset and gradual progression Disorder of expressivelanguage is the B) Non-fluent spontaneous speech with at dominantfeature initially and throughout the least one of the following:agrammatism, disease course. Other aspects of cognition are phonemicparaphasias or anomia intact or relatively well preserved.

The discovery that TDP-43-positive inclusions characterize ALS andFTLD-TDP (Neumann et al 2006) was quickly followed by the identificationof missense mutations in the TARDBP gene in both familial and sporadiccases of ALS (Gitcho et al 2008; Sreedharan et al., 2008). So far, 38different TARDBP mutations have been reported in 79 genealogicallyunrelated families worldwide (Mackenzie et al 2010). TARDBP mutationsaccount for approximately 4% of all familial and around 1.5% of sporadicALS cases.

As of December 2010, mutations in thirteen genes which are associatedwith familial and sporadic ALS have been identified. Linkage of ALS tofive other chromosome loci has been demonstrated but thus far specificmutations have not been identified.

Methylthioninium (MT) in TDP-43 Proteinopathies

MT has a mode of action which targets and can reduce TDP-43 proteinaggregation in cells, which is a pathological feature of the vastmajority of both familial and sporadic ALS and is also characteristic ofFTLD-P.

In addition laboratory data shows that methylthioninium inhibits theformation of TDP-43 aggregates in SH-SY5Y cells. Following treatmentwith 0.05 μM MT, the number of TDP-43 aggregates was reduced by 50%.These findings were confirmed by immunoblot analysis (Yamashita et al2009).

The compounds and compositions of the invention may therefore be usefulfor the treatment of amyotrophic lateral sclerosis (ALS) andfrontotemporal lobar degeneration (FTLD).

Methylthioninium (MT) in Huntington's Disease and PolyglutamineDisorders

MT can reduce polyglutamine protein aggregation in cells, which is apathological feature of Huntington's disease. Huntington's disease iscaused by expansion of a translated CAG repeat located in the N-terminusof huntingtin. Wild-type chromosomes contain 6-34 repeats whereas, inHuntington's disease, chromosomes contain 36-121 repeats. The age ofonset of disease correlates inversely with the length of the CAG tractsthat code for polyglutamine repeats within the protein.

Laboratory data shows that methylthioninium inhibits the formation ofaggregates of a huntingtin derivative containing a polyglutamine stretchof 102 residues in zebrafish (van Bebber et al. 2010). MT, when testedat 0, 10 and 100 μM, prevented the formation of such aggregates inzebrafish in a dose dependent manner.

The compounds and compositions of the invention may therefore be usefulfor the treatment of Huntington's disease and other polyglutaminedisorders such as spinal bulbar muscular atrophy (or Kennedy disease),and dentatorubropallidoluysian atrophy and various spinocerebellarataxias (Orr & Zoghbi, 2007).

Mitochondrial Diseases and Lafora Disease

The organ most frequently affected in mitochondrial disorders,particularly respiratory chain diseases (RCDs), in addition to theskeletal muscle, is the central nervous system (CNS). CNS manifestationsof RCDs comprise stroke-like episodes, epilepsy, migraine, ataxia,spasticity, movement disorders, psychiatric disorders, cognitivedecline, or even dementia (mitochondrial dementia). So far mitochondrialdementia has been reported in MELAS, MERRF, LHON, CPEO, KSS, MNGIE,NARP, Leigh syndrome, and Alpers-Huttenlocher disease (Finsterer, 2009).There are four complexes in the mitochondrial respiration chain,involving a series of electron transfers. Abnormal function of any ofthese complexes can result in mitochondrial diseases secondary to anabnormal electron transport chain and subsequent abnormal mitochondrialrespiration. Complex III of the mitochondrial respiration chain acts totransfer electrons to cytochrome c.

Compounds and compositions of the invention may also be used to treatmitochondrial diseases which are associated with a deficient and/orimpaired complex III function of the respiration chain. The compoundshave the ability to act as effective electron carrier and/or transfer,as the thioninium moiety has a low redox potential converting betweenthe oxidised and reduced form. In the event of an impaired and/ordeficient function of Complex III leading to mitochondrial diseases,compounds of the invention are also able to perform the electrontransportation and transfer role of complex III because of the abilityof the thioninium moiety to shuttle between the oxidised and reducedform, thus acting as an electron carrier in place of sub-optimallyfunctioning complex III, transferring electrons to cytochrome c.

Compounds and compositions of the invention also have the ability togenerate an active thioninium moiety that has the ability to divertmisfolded protein/amino acid monomers/oligomers away from the Hsp70ADP-associated protein accumulation and/or refolding pathways, andinstead rechannel these abnormal folded protein monomers/oligomers tothe pathway that leads directly to the Hsp70 ATP-dependentubiquitin-proteasome system (UPS), a pathway which removes thesemisfolded proteins/amino acid monomers/oligomers via the direct route(Jinwal et al. 2009).

Lafora disease (LD) is an autosomal recessive teenage-onset fatalepilepsy associated with a gradual accumulation of poorly branched andinsoluble glycogen, termed polyglucosan, in many tissues. In the brain,polyglucosan bodies, or Lafora bodies, form in neurons. Inhibition ofHsp70 ATPase by MT (Jinwal et al. 2009) may upregulate the removal ofmisfolded proteins. Lafora disease is primarily due to a lysosomalubiquitin-proteasomal system (UPS) defect because of a mutation ineither the Laforin or Malin genes, both located on Chromosome 6, whichresult in inclusions that may accelerate the aggregation of misfoldedtau protein. Secondary mitochondrial damage from the impaired UPS mayfurther result in a suppressed mitochondrial activity and impairedelectron transport chain leading to further lipofuscin and initiatingthe seizures that are characteristic of Lafora disease.

The MT moiety may disaggregate existing tau aggregates, reduce more tauaccumulating and enhance lysosomal efficiency by inhibiting Hsp70ATPase. MT may lead to a reduction in tau tangles by enhancing theubiquitin proteasomal system removal of tau monomers/oligomers, throughits inhibitory action on Hsp70 ATPase.

Thus compounds and compositions of the present invention may haveutility in the treatment of Lafora disease.

Disease Conditions Treated—Other Disease Conditions

In some embodiments, the disease condition is skin cancer.

In some embodiments, the disease condition is melanoma.

In some embodiments, the disease condition is a viral, bacterial orprotozoal disease condition.

In some embodiments, the (protozoal) disease condition is malaria.Treatment may be in combination with one or more antimicrobial agents,for example, chloroquine and/or atovaquone.

In some embodiments, the (viral) disease condition is caused byHepatitis C, HIV, or West Nile Virus (WNV).

Other Uses

Another aspect of the present invention pertains to use of a compound asdescribed herein, in a method of inactivating a pathogen in a sample(for example a blood or plasma sample), comprising the steps ofintroducing the compound into the sample, and exposing the sample tolight.

For example, in some embodiments, the method comprises the steps ofintroducing the compound into the sample, and then exposing the sampleto light.

Use as Ligands

The compounds described herein that are capable of inhibiting theaggregation of tau protein will also be capable of acting as ligands orlabels of tau protein (or aggregated tau protein). Thus, in someembodiments, the compound of the invention is a ligand of tau protein(or aggregated tau protein).

Such compounds (ligands) may incorporate, be conjugated to, be chelatedwith, or otherwise be associated with, other chemical groups, such asstable and unstable detectable isotopes, radioisotopes,positron-emitting atoms, magnetic resonance labels, dyes, fluorescentmarkers, antigenic groups, therapeutic moieties, or any other moietythat may aid in a prognostic, diagnostic, or therapeutic application.

For example, in some embodiments, the compound is as defined herein, butwith the additional limitation that the compound incorporates, isconjugated to, is chelated with, or is otherwise associated with, one ormore (e.g., 1, 2, 3, 4, etc.) detectable labels, for example, isotopes,radioisotopes, positron-emitting atoms, magnetic resonance labels, dyes,fluorescent markers, antigenic groups, or therapeutic moieties.

In some embodiments, the compound is a ligand as well as a label, e.g.,a label for tau protein (or aggregated tau protein), and incorporates,is conjugated to, is chelated with, or is otherwise associated with, oneor more (e.g., 1, 2, 3, 4, etc.) detectable labels.

For example, in some embodiments, the compound is as defined above, butwith the additional limitation that the compound incorporates, isconjugated to, is chelated with, or is otherwise associated with, one ormore (e.g., 1, 2, 3, 4, etc.) detectable labels.

Labelled compounds (e.g., when ligated to tau protein or aggregated tauprotein) may be visualised or detected by any suitable means, and theskilled person will appreciate that any suitable detection means as isknown in the art may be used.

For example, the compound (ligand-label) may be suitably detected byincorporating a positron-emitting atom (e.g., ¹¹C) (e.g., as a carbonatom of one or more alkyl group substituents, e.g., methyl groupsubstituents) and detecting the compound using positron emissiontomography (PET) as is known in the art.

Such ¹¹C. labelled compounds may be prepared by adapting the methodsdescribed herein in known ways, for example, in analogy to the methodsdescribed in WO 02/075318 (see FIGS. 11 a, 11 b, 12 therein) and WO2005/030676.

Thus, another aspect of the present invention pertains to a method oflabelling tau protein (or aggregated tau protein) comprising the stepof: (i) contacting the tau protein (or aggregated tau protein) with acompound that incorporates, is conjugated to, is chelated with, or isotherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels. The compound may be provided as a composition asdescribed herein.

Another aspect of the present invention pertains to a method ofdetecting tau protein (or aggregated tau protein) comprising the stepsof: (i) contacting the tau protein (or aggregated tau protein) with acompound that incorporates, is conjugated to, is chelated with, or isotherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels, and (ii) detecting the presence and/or amount of saidcompound bound to tau protein (or aggregated tau protein). The compoundmay be provided as a composition as described herein.

Another aspect of the present invention pertains to a method ofdiagnosis or prognosis of a tau proteinopathy in a subject believed tosuffer from the disease, comprising the steps of: (i) introducing intothe subject a compound capable of labelling tau protein or aggregatedtau protein, particularly tau protein (e.g., a compound thatincorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectablelabels); (ii) determining the presence and/or amount of said compoundbound to tau protein or aggregated tau protein in the brain of thesubject; and (iii) correlating the result of the determination made in(ii) with the disease state of the subject. The compound may be providedas a composition as described herein.

Another aspect of the present invention pertains to a compound capableof labelling tau protein or aggregated tau protein (e.g., a compoundthat incorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectablelabels), for use in a method of diagnosis or prognosis of a tauproteinopathy. The compound may be provided as a composition asdescribed herein.

Another aspect of the present invention pertains to use of a compound ofthe invention capable of labelling tau protein or aggregated tauprotein, particularly tau protein (e.g., a compound that incorporates,is conjugated to, is chelated with, or is otherwise associated with, oneor more (e.g., 1, 2, 3, 4, etc.) detectable labels), in a method ofmanufacture of a diagnostic or prognostic reagent for use in thediagnosis or prognosis of a tau proteinopathy. The compound may beprovided as a composition as described herein.

Those skilled in the art will appreciate that instead of administeringligands/labels directly, they could be administered in a precursor form,for conversion to the active form (e.g., ligating form, labelling form)by an activating agent present in, or administered to, the same subject.

The ligands disclosed herein may be used as part of a method ofdiagnosis or prognosis. It may be used to select a patient fortreatment, or to assess the effectiveness of a treatment or atherapeutic (e.g., an inhibitor of tau protein aggregation) administeredto the subject.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound of the invention, or a material, compositionor dosage from comprising said compound, which is effective forproducing some desired therapeutic effect, commensurate with areasonable benefit/risk ratio, when administered in accordance with adesired treatment regimen.

Similarly, the term “prophylactically effective amount,” as used herein,pertains to that amount of a compound of the invention, or a material,composition or dosage from comprising said compound, which is effectivefor producing some desired prophylactic effect, commensurate with areasonable benefit/risk ratio, when administered in accordance with adesired treatment regimen.

“Prophylaxis” in the context of the present specification should not beunderstood to circumscribe complete success i.e. complete protection orcomplete prevention. Rather prophylaxis in the present context refers toa measure which is administered in advance of detection of a symptomaticcondition with the aim of preserving health by helping to delay,mitigate or avoid that particular condition.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT,ADEPT, etc.); surgery; radiation therapy; and gene therapy.

For example, it may be beneficial to combine treatment with a compoundas described herein with one or more other (e.g., 1, 2, 3, 4) agents ortherapies.

The particular combination would be at the discretion of the physicianwho would select dosages using his/her common general knowledge anddosing regimens known to a skilled practitioner.

The agents (i.e., a compound as described herein, plus one or more otheragents) may be administered simultaneously or sequentially, and may beadministered in individually varying dose schedules and via differentroutes. For example, when administered sequentially, the agents can beadministered at closely spaced intervals (e.g., over a period of 5-10minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart,or even longer periods apart where required), the precise dosage regimenbeing commensurate with the properties of the therapeutic agent(s).

The agents (i.e., a compound as described here, plus one or more otheragents) may be formulated together in a single dosage form, oralternatively, the individual agents may be formulated separately andpresented together in the form of a kit, optionally with instructionsfor their use.

Routes of Administration

The compound of the invention, or pharmaceutical composition comprisingit, may be administered to a subject/patient by any convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal (including, e.g., intracatheter injection into the brain);by implant of a depot or reservoir, for example, subcutaneously orintramuscularly.

Preferred compositions are oral compositions, formulated as described inmore detail hereinafter.

The Subject/Patient

The subject/patient may be an animal, a mammal, a placental mammal, arodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., amouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine(e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine(e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate,simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), amonotreme (e.g. platypus), an ape (e.g., gorilla, chimpanzee,orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In some embodiments, the subject/patient is a human.

Compositions/Formulations

While it is possible for the compound of the invention to be used (e.g.,administered) alone, it is often preferable to present it as acomposition or formulation.

Another aspect of the invention therefore provides a compositioncomprising a compound as described herein, and a pharmaceuticallyacceptable carrier or diluent.

In some embodiments, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a compound asdescribed herein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

In some embodiments, the composition is a pharmaceutical compositioncomprising at least one compound, as described herein, together with oneor more other pharmaceutically acceptable ingredients well known tothose skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In some embodiments, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled compound, as defined herein, together with one ormore other pharmaceutically acceptable ingredients well known to thoseskilled in the art, e.g., carriers, diluents, excipients, etc. Ifformulated as discrete units (e.g., tablets, etc.), each unit contains apredetermined amount (dosage) of the compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound isdissolved, suspended, or otherwise provided (e.g., in a liposome orother microparticulate). Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the blood(or other relevant bodily fluid) of the intended recipient. Examples ofexcipients include, for example, water, alcohols, polyols, glycerol,vegetable oils, and the like. Examples of suitable isotonic carriers foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the compound in the liquid is from about 1 ng/ml to about 10 μg/ml,for example from about 10 ng/ml to about 1 μg/ml. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

EXAMPLES OF SOME PREFERRED FORMULATIONS

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising 20 to 300 mg of a compoundas described herein (e.g., obtained by, or obtainable by, a method asdescribed herein; having a purity as described herein; etc.), and apharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the dosage unit is a tablet.

In some embodiments, the dosage unit is a capsule.

In some embodiments, said capsules are gelatine capsules.

In some embodiments, said capsules are HPMC(hydroxypropylmethylcellulose) capsules.

In some embodiments, the amount is 30 to 200 mg.

In some embodiments, the amount is about 30 mg.

In some embodiments, the amount is about 60 mg.

In some embodiments, the amount is about 100 mg.

In some embodiments, the amount is about 150 mg.

In some embodiments, the amount is about 200 mg.

Throughout the present specification dosage amounts, e.g. as set outabove, may refer to the amount of the compound itself or may refer tothe amount of free base equivalent (i.e. the amount of LMT moiety)contained in the dosage unit. Both these alternatives are expresslydisclosed by the present invention.

In some embodiments, the pharmaceutically acceptable carrier, diluent,or excipient is or comprises one or both of a glyceride (e.g., Gelucire44/14®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicondioxide (e.g., 2% Aerosil 200®; Colliodal Silicon Dioxide PhEur, USP).

Novel Formulations—Solid Dosage Forms

Processes generally used for tablet formulation and film coating oftenrequire the use of heat accompanied by low humidity during the dryingprocess.

LMTM and the other leuco-methylthionium salts are potentially prone tooxidation to methylthioninium moiety (MT) and to degradation e.g. to LAzure B (LAB) (see Scheme, below):

For a material such as e.g. LMTM, which is prone to oxidation (asexplained above), conventional formulation processes may therefore leadto degradation and hence, potentially, to instability in the performanceof the product.

The principle behind the formulations of the present invention istherefore the provision of a method of manufacture of compressedpharmaceutical formulations and capsules containing leuco-methylthioniumsalts e.g. bis(methanesulfonate) (LMTM) as the active substance, bydirect tablet compression technology or by other unique tablettingtechniques, and by encapsulation, in which the active substance existssubstantially in a stable form.

The most commonly used method for the preparation of solid dosage formsis wet granulation (also called moist granulation). This involves addinga granulating fluid to a powder. The granulating fluid may be water orsome other solvent that is sufficiently volatile that can subsequentlybe removed by drying. The granulating fluid may also include a binder.Once the solvent has been removed, the resulting mass is milled.

Wet granulation is often preferred over direct compression because wetgranulation is more likely to overcome any problems associated with thephysical characteristics of various ingredients in the formulation. Wetgranulation provides material which has the required flow and cohesiveproperties necessary to obtain an acceptable solid dosage form. Thecontent uniformity of the solid dosage form is generally improved withwet granulation because all of the granules usually contain the sameamount of drug. Segregation of the drug from excipients is also avoided.

In direct compression, the individual constituents of the composition tobe compressed are mixed without previous granulation and then directlycompressed. Whilst this appears to be an elegant and simple process, itmay be difficult to obtain with it commercially usable tablets whichhave sufficient strength yet which also disintegrate sufficientlyrapidly after administration. Also, many active substances cannot beprocessed by direct compression since they cannot be compressed withouta granulation step.

It has now, surprisingly, been found that compounds of the presentinvention are stable in a dry compressed solid dosage form such as atablet, during manufacture and storage, and that the amount ofdegradation products such as L Azure B (LAB) and methylthioninium (MT)formed can be controlled within the specifications (for example, LABless than 2% and MT less than 12%).

This is in contrast to the behaviour of e.g. LMTM when processed byconventional wet granulation processes. Without wishing to be bound bytheory, in conventional wet granulation processes LMTM, for instance,may be very unstable and a substantial amount of LAB and MT may beformed.

Accordingly, one aspect of the present invention provides apharmaceutical composition comprising a compound of the invention, insolid dosage form. The composition preferably further comprises at leastone diluent suitable for dry compression. The pharmaceutical compositionis characterised in that the compound exists in a substantially stableform.

Another aspect of the invention provides a free-flowing, cohesivepowder, comprising a compound of the invention and at least one diluentsuitable for dry compression, and optionally one or more otherexcipients, said powder being capable of being compressed into a soliddosage form.

These compositions and formulations are initially described herein withrespect to the bis(sulfonate) salts of the present invention, inparticular LMTM. However, the advantages of the present formulationmethods are equally applicable to other members of theleuco-methylthionium family of salts

For example, the formulations described herein are applicable also tothe 3,7-diamino-10H-phenothiazinium salts disclosed in WO2007/110627(WisTa Laboratories Ltd), which were briefly discussed above. Theseinclude leuco-methylthionium bis(hydrobromide) (LMT.2HBr, LMTB) andleuco-methylthionium bis(hydrochloride) (LMT.2HCl, LMTC). Therefore, ina broader aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of the following formula I:

-   -   wherein:        -   R¹, R⁹, R^(3NA), R^(3NB), R^(7NA) and R^(7NB) are as            previously defined;        -   and wherein each of HX¹ and HX² is independently a protic            acid;            or a pharmaceutically acceptable salt, solvate, or hydrate            thereof;            in a solid dosage form as described herein.

For completeness, it is noted that, as would be understood by oneskilled in the art, the above formula could equally be written as:

wherein X¹ and X² are the corresponding counterions.

Preferably X¹ and X² are independently sulfonate (such as alkylsulfonateor arylsulfonate, for example R^(A)SO₃ ⁻ or R^(B)SO₃ ⁻ as defined above)or halide (Cl⁻, Br⁻, I⁻). In other words, HX¹ and HX² are preferablyindependently sulfonic acids (R^(A)SO₃H, R^(B)SO₃H) or hydrohalides(HCl, HBr, HI).

As used hereafter, the term ‘active ingredient’ refers to the relevantleuco(methylthioninium) salt. In other words it refers to a compound offormula (I), such as a compound of the invention, for example LMTM.

Another aspect of the invention provides a process for the manufactureof said pharmaceutical compositions, by a dry compression method. Theprocess preferably comprises dry compression of an intimate powdermixture of the active compound with at least one diluent suitable fordry compression, and optionally one or more other excipients.

In some embodiments, the process comprises direct compression.

In some embodiments, the process comprises simple direct compression.

In some embodiments, the process comprises dry granulation.

In some embodiments, the process comprises moist granulation ofexcipients, followed by addition of the active ingredientextra-granularly.

Solid dosage forms according to the invention advantageously exhibitlong-term chemical and physical stability of the active ingredient(compound of the invention—e.g. LMTM). The pharmaceutical compositionsaccording to the invention also have fast dissolution rates, even afterlong-term storage.

A “substantially stable” form of the active ingredient means, in thepresent context, a form which does not react to form impurities such asoxidative impurities or other degradation products to any significantextent during the formulation process, or on storage of the formulatedproduct.

Therefore, in the present context, it may refer to a material whichcontains, for example, less than 20% w/w, less than 15% w/w, or lessthan 10% w/w of oxidative impurities or other degradation products. Inother words the material contains at least 80% w/w, at least 85% w/w, orat least 90% w/w of the pure active ingredient, in its original(unreacted) form.

In some embodiments, the material containing the active ingredient maycontain, for example, less than 20% w/w, less than 15% w/w, less than12% w/w, or less than 10% w/w of MT. In some embodiments, the materialmay contain, for example, less than 5% w/w, less than 3% w/w, or lessthan 2% w/w of LAB.

A “stable” tablet is, in the context of the present invention, a tabletthat remains substantially stable after prolonged storage undercontrolled conditions of temperature and humidity. Stability testing maybe carried out with the solid dosage forms directly exposed to thechosen environmental conditions, or with the solid dosage formscontained within packaging.

Content of Active Ingredient

The amount of the active ingredient in the uncoated composition isgenerally more than about 10% w/w, but can be more than 20%, or morethan 30% w/w. The amount of the active ingredient is generally less thanabout 70% w/w, and usually less than 60% or less than 50% w/w in atablet formulation. Typically, the amount of the active ingredient inthe uncoated tablet core composition is thus from about 10% w/w (or 20%or 30%) to about 70% w/w (or 60% or 50%). Where a coating is applied tothe composition, as described below, the overall weight of thecomposition is increased and thus the percentage of the activeingredient in the overall composition is somewhat reduced.

Diluents

The active ingredient may not be inherently compressible and thus mayrequire addition of suitable diluents to aid compression.

The pharmaceutical compositions of the invention therefore commonlycomprise at least 15% w/w, more commonly at least 20%, at least 30%, atleast 40% or at least 50% w/w of diluent(s).

Diluents that may be used include one or more of microcrystallinecellulose, lactose, mannitol, calcium salts such as, calcium phosphatedibasic, calcium sulphate and calcium carbonate, and sugars such aslactose, sucrose, dextrose and maltodextrin.

Preferred diluents are microcrystalline cellulose, lactose and mannitol.Spray-dried forms of lactose and mannitol are particularly suitableforms of those compounds for direct compression or dry granulationtechniques.

It has unexpectedly been found that when an active ingredientasdescribed herein, for example a compound of the present invention, suchas LMTM, is formulated with dry compression diluents such as one or moreof microcrystalline cellulose, spray dried lactose, anhydrous lactoseand mannitol, the resulting solid dosage forms are stable in the sensethat the active ingredient remains chemically stable, even afterextended storage.

The invention thus provides a method of preparing low, medium- orhigh-dose tablets, for example low, medium-, or high-dose LMTM tablets,that are stable and have good dissolution profiles, acceptable degreesof hardness and resistance to chipping, as well as a shortdisintegration time.

Dissolution of Compositions of the Invention

The present inventors have also surprisingly found that the unique soliddosage forms described herein provide a very fast dissolution rate.

As explained hereinbefore, and without wishing to be bound by theory, itis thought that the active methylthioninium (MT) moiety may preferablybe absorbed from the stomach and/or the upper GI tract. Afast-disintegrating and fast-dissolving formulation of theleuco(methylthioninium) salts would therefore be advantageous, sincethis would deliver the maximum possible amount of drug to the intendedpoint of absorption.

The fast dissolution rate of the solid dosage forms described hereinmeans that they are capable of dissolving rapidly in the stomach and/orupper GI tract and hence presenting the active ingredient thereeffectively, for rapid absorption.

In some embodiments, the formulations of the invention, when evaluatedusing a standard pharmacopeial method, provide at least 80% dissolutionwithin 30 minutes, preferably at least 80% dissolution within 15minutes, more preferably at least 80% dissolution within 10 minutes.

In some embodiments, the formulations of the invention, when evaluatedusing a standard pharmacopeial method, provide at least 90% dissolutionwithin 30 minutes, preferably at least 90% dissolution within 15minutes, more preferably at least 90% dissolution within 10 minutes.

In some embodiments, the formulations of the invention, when evaluatedusing a standard pharmacopeial method provide at least 95% dissolutionwithin 30 minutes, preferably at least 95% dissolution within 15minutes, more preferably at least 95% dissolution within 10 minutes.

Dissolution rates may be measured by standard pharamcopeial methods asdescribed in United States Pharmacopeia (USP) General Chapter <711>. Thecurrent USP is USP 34 (2011). For example, dissolution rates for theformulations of the invention may be measured using apparatus accordingto USP Dissolution Apparatus 2 (Paddle).

In some embodiments, the dissolution rates above are evaluated in 0.1Mhydrochloric acid at a working concentration of ˜5 μg/ml LMT, withstirring at 50 rpm paddle speed. In some embodiments, the dissolutionrates are evaluated by spectrophotometric analysis. In some embodiments,analysis comprises UV/vis spectrophotometry (λ_(max LMT)=255 nm).

As a consequence of their surprisingly high dissolution rate, theformulation methods described herein can provide the active compoundwith a high degree of bioavailability.

The fast dissolution rate is maintained after prolonged storage, even ifstorage is under ‘stressed’ conditions (i.e. increased temperature andhumidity). The fast dissolution rate, and hence the goodbioavailability, of compositions formulated according to the processesof the present invention is also highly tolerant of variations in theformulation itself.

Other Ingredients

The pharmaceutical composition will generally also include a lubricant.Examples of lubricants include magnesium stearate, calcium stearate,sodium stearyl fumarate, stearic acid, glycerylbehaptate, polyethyleneglycol, ethylene oxide polymers (for example, those available under theregistered trademark Carbowax from Union Carbide, Inc., Danbury, Conn.),sodium lauryl sulphate, magnesium lauryl stearate, mixtures of magnesiumstearate with sodium lauryl sulphate, and hydrogenated vegetable oil.Preferred lubricants include calcium stearate, magnesium stearate andsodium stearyl fumarate. Most preferred as the lubricant is magnesiumstearate. Lubricants generally comprise from about 0.5 to about 5.0% ofthe total (uncoated) tablet weight. The amount of lubricant employed isgenerally from about 1.0 to about 2.0%, preferably 0.5 to 2.0% w/w.

In addition to the diluent(s) and lubricant(s), other conventionalexcipients may also be present in the pharmaceutical compositions of theinvention. Such additional excipients include disintegrants, binders,flavouring agents, colours and glidants. Some excipients can servemultiple functions, for example as both binder and tablet disintegrant.

A tablet disintegrant may be present in an amount necessary to achieverapid dissolution. Disintegrants are excipients which oppose thephysical forces of particle bonding in a tablet or capsule when thedosage form is placed in an aqueous environment. Examples ofdisintegrants include crosslinked polyvinylpyrrolidone (crospovidone),sodium starch glycolate, crosslinked sodium carboxymethyl cellulose(sodium croscarmellose), and pregelatinized starch. Generally the amountof disintegrant can be from 0 to about 25% w/w, more commonly from about1% to about 15% w/w, and usually less than 10% or less than 5% w/w, ofthe composition.

Binders are excipients which contribute to particle adhesion in a solidformulation. Examples of binders include cellulose derivatives(carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, microcrystallinecellulose) and sugars such as lactose, sucrose, dextrose, glucose,maltodextrin, and mannitol, xylitol, polymethacrylates,polyvinylpyrrolidone, sorbitol, pregelatinized starch, alginic acids,and salts thereof such as sodium alginate, magnesium aluminum silicate,polyethylene glycol, carrageenan and the like. Generally, the amount ofbinder can vary widely, eg from 0% to 95% w/w of the composition. Asnoted above, excipients may serve multiple functions. For instance, thetabletting diluent may also serve as a binder.

Glidants are substances added to a powder to improve its flowability.Examples of glidants include magnesium stearate, colloidal silicondioxide (such as the grades sold as Aerosil), starch and talc. Glidantsmay be present in the pharmaceutical composition at a level of from 0 toabout 5% w/w. Again, however, it should be noted that excipients mayserve multiple functions. The lubricant, for example magnesium stearate,may also function as a glidant.

Examples of colours that may be incorporated into the pharmaceuticalcompositions of the invention include titanium dioxide and/or dyessuitable for food such as those known as FD&C dyes and natural colouringagents. A colouring agent is unlikely to be used in the powder mixturethat is compressed in accordance with the aspects of the inventiondiscussed above, but may form part of a coating applied to thecomposition, as described below, in which case the colouring agent maybe present in the film coat in an amount up to about 2.0% w/w.

The tablet is desirably coated with a conventional film coating whichimparts toughness, ease of swallowing, and an elegant appearance to thefinal product. Many polymeric film-coating materials are known in theart. A preferred film-coating material is hydroxypropylmethylcellulose(HPMC) or polyvinyl alcohol-part hydrolysed (PVA). HPMC and PVA may beobtained commercially, for example from Colorcon, in coatingformulations containing excipients which serve as coating aids, underthe registered trademark Opadry. Opadry formulations may also containtalc, polydextrose, triacetin, polyethyleneglycol, polysorbate 80,titanium dioxide, and one or more dyes or lakes. Other suitablefilm-forming polymers may also be used, includinghydroxypropylcellulose, vinyl copolymers such as polyvinyl pyrollidoneand polyvinyl acetate, and acrylate-methacrylate copolymers. Use of afilm coating is beneficial for ease of handling and because a bluecoloured uncoated core may stain the inside of the mouth duringswallowing. Coating also improves light stability of the dosage form.

Coating of the tablets may conveniently be carried out using aconventional coating pan. In preferred embodiments of the process, thecoating pan is pre-heated using heated inlet air until the exhausttemperature reaches 35°-55° C., more preferably 40-50° C. This maytypically require application of heated inlet air at an inlettemperature of 45-75° C., preferably 50-65° C., for 10-15 minutes. Thetablet cores containing the active ingredient (e.g. LMTM) are then addedto the coating pan and the aqueous film coat applied. The spray rate iscontrolled such that the bed temperature is maintained at 38-48° C.,more preferably 42-44° C., until the desired weight gain (coatingweight) has been achieved.

Dry Compression Methods

‘Dry compression’, as used herein, refers to compression techniqueswhich do not involve the use of heat or moisture. Dry compression maycomprise direct compression of the active ingredient with suitablediluents or it may comprise dry granulation (for example slugging/doublecompression method or roller compaction).

Direct compression may comprise simple direct compression of the activeingredient with diluents suitable for direct compression. Alternativelyit may comprise granulation, for example moist granulation, of theexcipients to produce a dry granular excipient mixture which can then bedirectly compressed with the dry active ingredient (and optionallyfurther dry excipients). This may be referred to as ‘extra-granularincorporation’ of the active ingredient.

Accordingly, in some embodiments the solid dosage forms of the inventionmay be produced in a manufacturing process which comprises simple directcompression. In this embodiment, the tablet ingredients, i.e. the activeingredient (e.g. LMTM), diluent(s) and other optional excipients, areblended together in solid particulate form to create an intimatemixture, e.g. in a tumbling blender, and are then compressed using atablet machine.

In other embodiments, the composition is prepared by a dry granulationprocess. Dry granulation refers to the process of granulating withoutthe use of granulating fluids. In order for a material to bedry-granulated, at least one of its constituents, either the activeingredient or a diluent, must have cohesive properties. Dry granulationmay be performed by a process known as “slugging”. In “slugging”, thematerial to be granulated is first made into a large compressed mass or“slug”, typically using a tablet press with large flat-faced tooling (anexample of a linear press is illustrated in U.S. Pat. No. 4,880,373). Afairly dense slug may be formed by allowing sufficient time for the airto escape from the material to be compacted. Compressed slugs are thenmilled through a desired mesh screen manually or automatically as, forexample, by way of a comminuting mill. Formation of granules by“slugging” is also known as precompression. When tablets are made fromthe granulated slugged material, the process is referred to as the“double compression method”.

Dry granulation may also be performed using a “roller compactor”. In aroller compactor, material particles are consolidated and densified bypassing the material between two high-pressure rollers. The densifiedmaterial from a roller compactor is then reduced to a uniform granulesize by milling. The uniform granules may then be mixed with othersubstances, such as a lubricant, to tablet the material (as, forexample, by way of a rotary tabletting machine). In addition topharmaceutical use, roller compaction is used in other industries, suchas the food industry, animal feed industry and fertilizer industry.

Dry granulation is nowadays generally understood to mean rollercompaction or slugging, and is well known to those skilled in the art(see, for instance, Pharmaceutical Dosage Forms: Tablets (Lieberman,Lachman, and Schwartz (Eds); Marcel Dekker, Inc, 2nd Edition, 1989) andRemington's Pharmaceutical Sciences (A. R. Gennaro (Ed); Mack PublishingCo, Easton, Pa., 18th edition, 1990)).

In further embodiments of the invention, tablets are prepared by moistgranulation of excipients and incorporation of the active ingredient(e.g. LMTM) extra-granularly. Typically, such a process involves wetmassing diluents such as lactose and/or microcrystalline cellulose withwater, optionally with the addition of a binder such as polyvinylpyrrolidone. The wet mass is dried, then passed through a mesh, to formgranules. The active ingredient and any remaining excipients, such as alubricant, are then blended with the dry granules and compressed to formtablets.

Use of Acids in the Compositions of the Invention

Compositions containing leuco(methylthioninium) compounds, includingcompounds of the invention such as LMTM may, in some embodiments, bestabilised by addition of an appropriate amount of certain acids to thebulk substance prior to formulation. These acids may be used to preventthe formation of further MT, both during formulation and throughout thelife of the product, thereby providing a stable pharmaceuticalcomposition for the purposes of obtaining regulatory approval withassociated cost savings in packaging.

According to the present invention, therefore, there is also provided apharmaceutical composition comprising an active ingredient as describedherein and a pharmaceutically acceptable carrier, characterised in thatsaid formulation additionally comprises an acid in an amount sufficientto prevent the formation of MT. Without wishing to be bound by theory,it is thought that acids having a pK1 of greater than 1.5 are preferred.In some embodiments, the acid is present in an amount of from 5% to 25%w/w.

Preferably the composition is prepared by a dry compression method asdescribed above.

Preferred acids for the purposes of the invention are maleic acid (pK11.9), phosphoric acid (pK1 2.12), ascorbic acid (pK1 4.17), sorbic acid(pK1 4.76), aspartic acid, and sialic acid. The stabilising effect ofthe added acid may be enhanced by the selection of an appropriatecarrier. The carrier is preferably mannitol, a cellulosic material, or astarch, or mixtures thereof. The carrier is typically present in anamount of at least 40% w/w of the formulation.

Particle Size

It has also been found that a significant reduction in the formation ofMT can also be achieved by the selection of an appropriate particle sizerange for the dry powder blend, typically wherein more than 10% of theparticles have a size greater than 10 microns. Therefore according toanother aspect of the invention, there is provided a pharmaceuticalcomposition comprising an active ingredient as described herein and apharmaceutically acceptable carrier, additionally characterised in thatsaid composition comprises particles of which more than 10% have a sizegreater than 10 microns.

Carriers

It has been found that a significant reduction in the formation of MTcan also be achieved by the choice of an appropriate carrier,particularly one having a particle shape which is averse to the entry ofwater. Elcema TM, for example, which has long, lamellar particles whichare smooth and flat in shape with a non-porous surface, appears toreduce MT formation by limiting the access of water. Ethylcellulose,mannitol and Starch 1500 TM and Microcrystalline cellulose are alsoparticularly suitable for this purpose.

Therefore according to another aspect of the invention, there isprovided a pharmaceutical composition comprising aleuco(methylthioninium) compound, for example a compound of theinvention such as LMTM, and a pharmaceutically acceptable carrier,characterised in that said carrier is Elcema TM, ethylcellulose,mannitol, or Starch 1500 TM.

Encapsulation

Stabilised dry powder blends in accordance with the invention may beformulated, for example, by compressing into tablets or filling intocapsules (with or without prior conversion to a granulated powder bymeans such as described in Formulation Examples 1 to 4) to givepharmaceutical compositions having excellent shelf life.

Capsules according to the invention are typically of gelatin orpreferably HPMC. Preferred excipients include lactose, starch, acellulose, milk sugar and high MW polyethylene glycols.

Conclusions

Pharmaceutical compositions and formulations prepared according to themethods described above are more stable, immediately after completion ofmanufacture, than formulations produced using conventional aqueousgranulation. Furthermore they can demonstrate enhanced stability onstorage.

For example, a pharmaceutical formulation thus prepared, with a contentof 10 to 50% by weight of LMTM, preferably 15% to 40% by weight of LMTM,makes it possible that in standard stability tests, for example in longterm accelerated stability testing, at a temperature of 25° C. and arelative humidity 60±5% the content of L Azure B does not increase bymore than 2%, relative to LMTM peak area, within a period of 24 months.

During processing and on storage leuco(methylthioninium) compounds, suchas LMTM may also oxidise to produce a small amount of MT (see Scheme,above).

The presence of relatively small concentrations (e.g. less than 12%) ofMT in the leuco-formulations of the present invention, althoughundesirable, is not considered to have adverse clinical significance perse as even if the body is presented with MT in its charged or oxidizedform from LMTM and the various other leuco salts, this can then bereduced to the uncharged (reduced) MT form prior to absorption. Inaddition to the small amount of MT formed during processing such asblending and tabletting, leuco-methylthioninium salts of the presentinvention may react with oxygen absorbed on the excipients and presentwithin the tablet to give more of the MT particularly in the presence ofmoisture.

One advantage of the formulations of this invention is to minimise theamount of MT formed in the tablets, e.g. to less than 12% over 2 yearswhen stored at 25° C. at a relative humidity of 60%. This refers to thecumulative amount of MT formed during both processing and storage of thetablets: generally, the formulation methods of the invention result inless than 5% MT formation during processing; a maximum of around 5-7% MTis then formed during storage of the finished pack. This provides ashelf-life of at least 24 months.

This is demonstrated in the Formulation Examples, below.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the compound, and compositions comprising the compound, canvary from patient to patient. Determining the optimal dosage willgenerally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the compound is in the range of about 100ng to about 25 mg (more typically about 1 μg to about 10 mg) perkilogram body weight of the subject per day.

In some embodiments, the compound is administered to a human patientaccording to the following dosage regime: about 100 mg, 3 times daily.

In some embodiments, the compound is administered to a human patientaccording to the following dosage regime: about 150 mg, 2 times daily.

In some embodiments, the compound is administered to a human patientaccording to the following dosage regime: about 200 mg, 2 times daily.

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit its scope.

Example 1 Synthesis and Characterisation Laboratory synthesis of10-Acetyl-N,N,N′,N′-tetramethylphenothiazine-3,7-diamine

Synthesis of 3,7-dinitro-10H-phenothiazine (2)

To a 3 necked 1 litre round bottom flask (RBF) fitted with athermometer, dropping funnel and a condenser was added phenothiazine (MW199.28 g/mol, 25.00 g, 125.5 mmol) and dimethylsulfoxide (250 ml) themixture was stirred for 2 minutes or until the phenothiazine dissolved.The condenser was then connected to a Dreschel bottle half filled withwater. Sodium nitrite (MW 69.00 g/mol, 51.94 g, 752.7 mmol) was thenadded to the RBF and acetic acid (150 ml) was added to the droppingfunnel. The acetic acid was then added to the RBF in a drop-wise fashionover a 20 minute period. The light yellow slurry becomes red in colourand a solid precipitated out of solution. Upon completion of the aceticacid addition the mixture was stirred for 2 hours at ambient temperature(36-20° C.) before increasing the temperature to 95° C. and stirring for17 hours. After this time the mixture was cooled to 50° C. and methanol(100 ml) was added and the mixture cooled further to 22° C. The cooledmixture was then filtered and the cake washed with methanol (3×25 ml).The washed cake was left on the filter with the vacuum applied for 30minutes before being dried for 15 hours at 50° C. to give the product asa brown solid (MW 289.27 g/mol, 29.45 g, 81%).

Notes

-   1. The addition of acetic acid produced NO_(x) gases, which was    converted to nitric acid by allowing the gas to bubble into a    Dreschel bottle half filled with water.-   2. The addition of acetic acid is exothermic and the mixture rises    from 22° C. to 36° C.-   3. Methanol was added to help dissolve any sodium acetate and as an    anti-solvent to maximise the product yield.-   4. The synthesis was also successful using dimethylformamide (DMF),    acetonitrile (MeCN), tetrahydrofuran (THF), acetone or    dimethoxyethane (DME) as the reaction solvent.

NMR: The product (5 mg) was dissolved in DMSO-d6 (1.5 ml) and mayrequire to be warmed to completely dissolve the solid.

δ_(H)(400 MHz; DMSO-d6): 6.72 (2H, d, J 8.8, ArH), 7.77 (2H, d, J 2.8,ArH), 7.87 (2H, dd, J 2.8, 8.8, ArH)

Synthesis of 3,7-dinitro-10-acetylphenothiazine (3)

To a 3 necked 500 ml round bottom flask fitted with a thermometer and acondenser was added 3,7-dinitro-10H-phenothiazine (MW 289.27 g/mol,29.00 g, 100 mmol), dimethylformamide (58 ml), acetic anhydride (MW102.09 g/mol, 102.09 g, 1000 mmol) and triethylamine (MW 101.19 g/mol,40.88 g, 401 mmol). The mixture was heated to 105° C. and stirred atthis temperature for 3 hours. The mixture was cooled to ambienttemperature (21° C.) before being cooled to 5° C. whereby it was stirredfor 1 hour. The product was isolated by filtration and washed withmethanol (3×30 ml) to give a light yellow crystalline solid, which wasdried at 50° C. for 15 hours (MW 331.31 g/mol, 26.94 g, 81%).

Notes

-   1. Crystals of the product form during the reaction, after ˜1 hour    at 105° C.-   2. Upon cooling the bulk of the product precipitates at ˜70° C.-   3. Product was orange in colour before it was washed with methanol.

NMR: The product (10 mg) was dissolved in DMSO-d6 (1.5 ml).

δ_(H)(400 MHz; DMSO-d6): 2.25 (3H, s, CH₃), 7.92 (2H, d, J 8.8, ArH),8.28 (2H, dd, J 8.8, 2, ArH), 8.47 (2H, d, J 2, ArH)

Synthesis of 10-Acetyl-N,N,N′N′-tetramethylphenothiazine-3,7-diamine (5)

To a 3 necked 100 ml round bottom flask fitted with a thermometer and acondenser was added 3,7-dinitro-10-acetylphenothiazine (MW 331.31 g/mol,5 g, 15.09 mmol), palladium on carbon (10%, dry, 0.5 g) and2-methyltetrahydrofuran (25 ml). The flask was evacuated and purged withhydrogen 5 times before the mixture was heated to 56° C. After 17 hoursthe reduction was judged to have reached completion (see tlc conditions)giving compound 4, and formalin was added (MW 30.03 g/mol, 14.7 g, 181.1mmol). The flask was once again evacuated and purged 5 times withhydrogen. After 71 hours from the addition of formalin (total time 88hours) at 56° C. the tetra-methylation was judged to be complete by tlc.The mixture was filtered at 50° C., the grey catalyst was washed with2-methyltetrahydrofuran (3×5 ml) and the filtrate and washings werecombined. To this solution was added methanol (5 ml) to homogenise themixture. Cooling to 5° C. resulted in a colourless solid precipitatingfrom solution. A further two volumes of methanol (10 ml) were added andthe slurry was stirred for 50 minutes at 5° C. The crude product wasisolated by filtration to give a colourless solid, which was washed withmethanol (3×5 ml) and dried at 50° C. for 16 hours (MW 327.45 g/mol,2.26 g, 46%). The filtrate from the isolation process had water added(50 ml), which gave further solid. The suspension was stirred at 5° C.for 2 hours before being collected by filtration, washed with methanol(3×5 ml) and dried at 50° C. for 13 hours. (MW 327.45 g/mol, 0.83 g,17%). The total yield of product was (3.09 g, 63%).

Notes

-   1. Normal phase tlc conditions, eluent 75% ethyl acetate, 25%    petroleum spirit (40-60° C.), and UV lamp at 254 nm.-   2. The retention factor of the dinitro starting material is 0.68 as    a yellow spot, the retention factor of the hydrogenation product is    0.25 as a blue spot and the retention factor of the methylation    product is 0.67 as a light blue spot.-   3. The method for the tlc analysis of the hydrogenation step was    direct spotting whereas the analysis of the methylation product had    water added to a reaction aliquot which was extracted with ethyl    acetate and then spotted.-   4. After 17 hours the tlc shows two spots, the major spot was the    reduction product the minor spot is unknown.-   5. After 88 hours the tlc shows mainly the tetra-methylated product    as the major spot.-   6. Typically the reduction and methylation would be complete within    72 hours.-   7. 1H NMR spectroscopy of the two samples gave identical spectra,    traces of 2-methyltetrahydrofuran were detected along with an    unknown signal at 5 ppm.

NMR: The product (10 mg) was dissolved in CDCl₃ (1.5 ml).

δ_(H)(400 MHz; CDCl₃): 2.09 (3H, s, CH₃), 2.86 (12H, s, NCH₃), 6.54 (2H,d, J 8, ArH), 6.64 (2H, s, ArH), 7.19 (2H, brd s, ArH)

Alternative Synthesis of10-Acetyl-N,N,N′N′-tetramethylphenothiazine-3,7-diamine (5)

To a 50 ml round bottom flask was added3,7-dinitro-10-acetylphenothiazine (MW 331.31 g/mol, 1 g, 3.02 mmol),zinc dust (MW 65.39 g/mol, 1.38 g, 21.13 mmol), methanol (6 ml) andtetrahydrofuran (2 ml). The mixture was heated to 50° C. after which awarm solution (45-50° C.) of aqueous ammonium chloride (MW 53.49 g/mol,2.26 g, 42.26 mmol dissolved in 6 ml of water) was added slowly tomaintain a gentle reflux. The mixture was then heated to 70° C. andstirred at this temperature for two hours after which it was cooled toambient temperature (23° C.). The cooled mixture was filtered to removethe zinc salts and the filtrate containing compound 4 was treated withparaformaldehyde (MW 30.03 g/mol, 1.09 g, 36.22 mmol), sodiumcyanoborohydride (MW 62.84 g/mol, 1.14 g, 18.11 mmol) and acetic acid (2ml). The mixture was heated to 50° C. and stirred at this temperaturefor 3 hours. After cooling to ambient temperature (23° C.), water (2×10ml) was added and the colourless slurry was stirred for 16 hours. Thesolid was then collected by filtration and washed with methanol (3×2 ml)to give the title compound (MW 327.45 g/mol, 0.91 g, 92%) as anoff-white solid.

Notes

-   1. Reduction reaction using zinc and aqueous ammonium chloride was    fast and clean taking only 2 hours to reach completion with no other    spots recorded by tlc analysis.-   2. The reductive methylation using sodium cyanoborohydride,    paraformaldehyde and acetic acid was fast and clean, taking only 3    hours to reach completion.

NMR: The product (10 mg) was dissolved in CDCl₃ (1.5 ml).

δ_(H)(400 MHz; CDCl₃): 2.17 (3H, s, CH₃), 2.94 (12H, s, NCH₃), 6.61 (2H,d, J 8, ArH), 6.71 (2H, s, ArH), 7.26 (2H, brd s, ArH)

Synthesis 1 Synthesis ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumbis(methanesulphonate) (LMT.2MsOH)

10-Acetyl-N,N,N′N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) (150g) was added to a 3-neck round bottomed flask. Toluene (1.8 l) was addedand the mixture heated to reflux for 30 min. The solution was allowed tocool to 70° C. before being passed through an in-line 5μ filter to ajacketed vessel fitted with distillation apparatus.¹ Toluene (150 ml)was added to the round bottom flask. This was used to rinse the transferline and filter. Approximately 1.4 l. of toluene was distilled off underreduced pressure.² The temperature was lowered to 18° C. before water(42 ml) was added.³ This was followed by the addition ofmethanesulphonic acid (MSA) (65.5 ml, 99%, 2.2 equiv.) over a 5 min.period.⁴ A second portion of water (18 ml) was added. The mixture washeated to 85° C. for 3 h by which time the reaction was judged completeby tlc analysis. The biphasic solution was allowed to cool to 50° C.before absolute EtOH (150 ml) was added over 20 min.⁵ The mixture wasseeded using 150 mg ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumbis(methanesulphonate).⁶⁻⁸ A second portion of EtOH (600 ml) was addedover 90 min.⁹ and the reaction allowed to cool to 20° C. over 1 h.¹⁰ Itwas stirred at this temperature for 1 h. before the solid was collectedby filtration. The cake was washed with 3×300 ml of MeCN,¹¹ sucked dryfor 5 min. and placed under vacuum overnight to give the product as ayellow crystalline solid (85-90% yield).

μ_(max) (KBr)/cm⁻¹; 3430 (NH), 3014 (═CH), 2649 (C—H), 1614 (C═C), 1487(C—C), 1318 (S═O), 1199 (SO2-O), 1059 (S═O), 823 (ArC═—H)

δ_(H) (600 MHz; CD₃OD); 2.71 (6H, s, SCH₃), 3.21 (12H, s, NCH₃), 6.75(2H, d, J=8.8 Hz, ArH), 7.22 (4H, d J 2.9 Hz, ArH), 7.24 (4H, dd J 2.9,8.8 Hz, ArH),

δ_(c) (100 MHz; CD₃OD); 38.2 (SCH₃), 45.9 (NCH₃), 115.0 (CH), 118.2(CH), 118.7 (QC), 119.9 (QH), 137.1 (CH), 142.8 (QC)

MP: 271° C.

m/z (EI+): Calculated 285.129970; Observed 285.131292 (100%, [M−2MSA]⁺).

m/z (ES−): Calculated 95; Observed 95 (100%, [M−LMT]⁻).

Elemental analysis % (C18H27N3O6S3): Calculated C (45.26), N (8.80), S(20.14), H (5.70); Observed C (45.19), N (8.76), S (19.84), H (5.53)

Notes

-   1. Heating to reflux ensures complete dissolution of AcMT for    transfer through 5μ filter. Toluene is a good solvent and a 70° C.    target is a compromise between ensuring the material stays in    solution and minimising potential damage to plastic transfer hoses    and filter.-   2. 500 ml of remaining toluene ensures reaction volume meets minimum    stir depth of reactor.-   3. Volume of water is controlled to ensure product crystallises out    as a free flowing precipitate. Seeding the reaction reduces the    impact of small variations in water volume.-   4. 2.2 equivalents of MSA are used to effect the hydrolysis and form    the salt whilst leaving a sufficient quantity of excess acid (0.2    equiv.) to ensure the stability of the product in solution. Addition    of MSA causes a slight exotherm, hence the 5 minute addition time.-   5. EtOH is used as counter solvent to precipitate the product. A    portion is added before the seed to ensure the seed does not    dissolve. An extended addition time ensures controlled    crystallisation of the product (see notes 7 and 8).-   6. It is possible to carry out the reaction without the use of a    seed, however its incorporation ensures the early precipitation of    LMT.2MsOH which in turn prevents formation of by-products (such as    the alcohol ester EMS a potential genotoxic by-product—not detected    in the synthetic process) and encapsulation of EtOH.-   7. The seed is also useful as a means of controlling the particle    size of the product. When the seed material was used which has been    ground in a mortar and pestle to <100 μm a significant reduction in    the average particle size of the product is observed. When <100μ    seed which had not been ground was used no such effect was observed.    Therefore, without wishing to be bound by theory, it appears that    the ability of the seed to control the particle size is not a    function of the seed particle size, it is linked to the proportion    of internal or ‘new’ crystal faces that the crushing of the seed has    exposed.-   8. Finally, when the seed material was relatively large and    uncrushed a considerable amount of product (skin) may adhere to the    side of the reactor vessel during the EtOH addition. This may be    reduced by introducing a heat/cool cycle into the process after the    EtOH addition. However, an unexpected bonus of the utilisation of    the crushed seed was that the level of skinned material present    after EtOH addition was reduced by ˜90%. Therefore it was no longer    necessary to carry out the heat/cool cycle. It seems that this is    linked to the small seed size rather than new faces because when the    reaction was carried out using uncrushed <100μ seed the same    reduction in skinning was observed.-   9. The rate of EtOH addition has an effect on particle size and EtOH    inclusion. Fast addition (<1 h) reduces particle size however EtOH    inclusion increases. A slow addition (2 h) has the opposite effect    hence a balance must be struck.-   10. Rate of cooling has a similar although reduced effect. Fast cool    down (<1 h) leads to reduction in particle size with a concomitant    increase in EtOH levels. A slow cool has the opposite effect.-   11. EtOH is equally effective as MeCN at removing the related    substances, however its use is accompanied by a slight increase in    the level of retained EtOH.

Characterisation ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumbis(methanesulphonate) (LMT.2MsOH)

Elemental Analysis (Microanalysis)

The analysis has good correlation between the theoretical values and theanalysis values for carbon, nitrogen, hydrogen and sulphur.

Results of the elemental analysis:

Molecular Formula C₁₈H₂₇N₃O₆S₃ Element % Theoretical % Found C 45.2645.19 H 5.70 5.53 N 8.80 8.76 S 20.14 19.84¹H—Nuclear Magnetic Resonance (NMR) Spectroscopy

The ¹H NMR spectrum was obtained in deuterated methanol CD₃OD, on aVarian 600 MHz instrument and is shown in FIG. 1.

Assignment of the ¹H NMR spectrum is below:

Chemical Assignment Shift (ppm) Protons Group 15/16 2.71 6H, s 2 × SCH₃11/12/13/14 3.21 12H, s 2 × N(CH₃)₂ 1/9 6.75 2H, d, 8.8 Hz 2 × C—H(Aromatic) 4/6 7.22 2H, d, 2.9 Hz 2 × C—H (Aromatic) 2/8 7.24 2H, dd,8.8 and 2.9 Hz 2 × C—H (Aromatic)¹³C—Nuclear Magnetic Resonance (NMR) Spectroscopy

The ¹³C NMR spectrum was obtained on a Varian 400 MHz NMR instrument ata frequency of 100.56 MHz in deuterated methanol CD₃OD and is shown inFIG. 2.

The initial assignment of the ¹³C-NMR spectrum was based on correlationwith charts of known chemical shifts, (Literature Reference: StructureDetermination of Organic Compounds: Tables of Spectral Data, Pretsch E.,et al., Springer, London, p 122).

Further assignments utilised DEPT-135, HSQC and HMBC experiments tounambiguously confirm the assignments. DEPT-135 (DistortionlessEnhancement by Polarisation Transfer), HSQC (Heteronuclear SingleQuantum Coherence) and HMBC (Heteronuclear Multiple Bond Correlation)spectra were obtained on a Varian 400 MHz NMR instrument at a frequencyof 100.56 MHz (see FIGS. 3-5).

Assignment Chemical shift (PPM) NMR Region DEPT-135 15/16 38.2 Alkyl CH₃11/12/13/14 45.9 Alkyl CH₃ 1/9 115.0 Aromatic - C CH 4/6 118.2Aromatic - C CH 4a/5a 118.7 Aromatic - C C 2/8 119.9 Aromatic - C C 3/7137.1 Aromatic - C CH  9a/10a 142.8 Aromatic - C CInfrared Spectroscopy (IR)

A sample was thoroughly mixed and ground in a mortar and pestle with 200mg of anhydrous KBr. This mixture was then pressed into a disc, using adie at a pressure of 1500 psi. The IR spectrum was then obtained on aNicolet Avatar 320 FT-IR spectrometer. The spectrum is shown in FIG. 6.

Assignment of the infrared spectrum:

Peak Wavenumber (cm⁻¹) Peak Type Assignment ~3430 broad N—H stretch 3014medium ═C—H stretch 2649 medium C—H stretch 1614 medium C═C stretch 1487strong C—C stretch 1318 strong S═O stretch 1199 strong SO₂—O stretch1059 strong S═O stretch 823 strong Aromatic C—H stretchMass Spectrometry (MS)

Mass spectroscopic analysis was carried out using a Waters, LCT PremierXE mass spectrometer. A flow rate of 1 ml/hr was adopted. The sourceused for the analysis of the active component was electron impactionisation in the positive mode. The source used for the analysis of themethanesulphonate counter ion was electrospray ionisation in thepositive mode.

Using electron impact ionisation a major peak is observed at 285 (seeFIG. 7). This corresponds to the molecular ion C₁₆H₁₉N₃S. A comparisonof the exact mass measured and the theoretical value is provided below:

Theoretical Peak m/z Abundance (%) Assignment 285.129970 285.131292 100C₁₆H₁₉N₃S

The measured accurate mass is in good agreement with the calculated massfor C₁₆H₁₉N₃S.

Using electrospray ionisation a major peak is observed at 95 (see FIG.8). This corresponds to the molecular ion of the counter ion CH₃O₃S:

Peak m/z Abundance (%) Assignment 95 100 CH₃O₃SUltraviolet-Visible Spectroscopy (UV-Vis)

A 5 mg sample was dissolved in de-ionised water, and made up to 100 mlin a volumetric flask. The analysis was carried out using quartz curvetsin a Perkin Elmer Lambda 25 UV/Vis is spectrometer. The UV-Vis spectrumis shown in FIG. 9.

Assignment of the UV-Vis spectrum:

λ_(max) (nm) Absorbance 226 1.7615 255 3.5860 332 0.7527 664 0.1845

The extinction coefficient ε for the lambda max at 255 nm was 34254.64.This was calculated according to the Beer-Lambert Law:

$ɛ = \frac{A}{C \times l}$where A=Absorbance Log (l₀/l) 3.5860; C=Concentration Mol/L; I=pathlength 1 cmHigh Performance Liquid Chromatography (HPLC)

A 100 mg sample was submitted for HPLC analysis. The analysis wascarried out on an Agilent 1200 series with VWD Detector or PDA foridentity, according to the method summarised in the table below.

HPLC method:

Parameters Conditions Column Zorbax SB-CN, 50 × 4.6 mm, 3.5 μm. Columntemperature 283 K Mobile phase A: 0.1% v/v Formic acid in water B: 100%acetonitrile Flow rate 1 ml/min Injection volume 5 μl Stop time 22 min.Wavelength UV at 255 nm Bandwidth at 4 nm. Reference wavelength set atoff. PDA scan 190 nm to 800 nm (Identity only) Auto sampler 278 KProtected from light. temperature Time Mobile Phase Mobile Phase (min.)A (%) B (%) Mobile Phase Gradient 0.0 100 0 10.0 90 10 17.0 50 50 18.050 50 18.1 100 0 22.0 100 0

The HPLC trace is shown in FIG. 10. The organic purity was found to be99.45% w/w.

HPLC Analysis (% Purity) including retention times LMT MT⁺ Leuco Azure B6.39 min. 14.38 min. 5.77 min. 99.45 0.55 <0.05Crystalline Form

In the above-described method, LMT.2MsOH is produced in crystallineform. The crystalline form of LMT.2MsOH is illustrated by the X-raypowder diffraction spectrum shown in FIG. 11. The XRPD exhibits sharpsignals, indicative of a high degree of crystalline order. Variations inrelative peak intensity may be observed, which are attributable toorientation effects in combination with differences in particle size.Only slight variations in relative peak intensity (less than 50%) areobserved as a function of sample thickness (0.1 mm vs. 1.0 mm).

The crystal form is further characterised by FT-Raman, thermogravimetric(TG), differential scanning calorimetric (DSC), dynamic vapour sorption(DVS) analysis, and microscopy (FIGS. 12-16). This form may convenientlybe referred to as ‘Form A’.

Crystals for single crystal X-ray analysis were obtained from ethanol,methanesulfonic acid and water. See FIG. 17 c.

Instrumental Details

X-Ray Powder Diffraction: Bruker 08 Advance, Cu Ka radiation (λ=1.54180Å), 40 kV/40 mA, LynxEye detector, 0.02° step size in 2θ, 37 s per step,2.5°-50° 2θ scanning range. The samples were prepared on silicon singlecrystal sample holders with 0.1 or 1.0 mm depth without any specialtreatment other than the application of slight pressure to get a flatsurface. All samples were rotated during the measurement.

Differential Scanning Calorimetry: Perkin Elmer DSC 7. Gold cruciblesclosed under N₂, heating rate 20° C./min, scan from −50° C. to 280° C.

Dynamic Vapor Sorption: Projekt Messtechnik SPS 11-100n water vaporsorption analyzer. The samples were placed in aluminum crucibles on topof a microbalance and were equilibrated at 25° C. and 50% r.h. beforestarting a pre-defined humidity program at 25° C. (50-0-95-50% r.h.,scanning with Δ r.h.=5% h⁻¹ and with ‘isohumid’ equilibration periods atthe extreme values).

FT-Raman Spectroscopy: Bruker RFS100. Nd:YAG 1064 nm excitation, 50 mWlaser power, Ge-detector, 128 scans, range 50-3500 cm⁻¹, 2 cm⁻¹resolution. Aluminum sample holder.

Polarizing Light Microscopy: Leitz Orthoplan microscope with LeicaOFC280 CCO camera.

TG: TA Instruments TGA Q5000. Open aluminum crucible, N₂ atmosphere,heating rate 10° C. min⁻¹, range 25-300° C.

TG-FTIR: Netzsch Thermo-Microbalance TG 209 with Bruker FT-IRSpectrometer Vector 22. Aluminum crucible with micro-hole, N2atmosphere, heating rate 10° C. min⁻¹, range 25-250° C.

Without wishing to be bound by theory, it is suggested that this formrepresents the only stable polymorphic form of LMT.2MsOH. Polymorphismstudies have shown that Form A is reproduced in nearly allcrystallisation systems (studies were performed using de-gassedsolvents, under an inert atmosphere).

Amorphous LMT.2MsOH can be prepared by evaporation of an aqueoussolution of LMT.2MsOH, however the amorphous material recrystallises toForm A upon further drying.

Industrial Scale Synthesis of AcMT and LMT.2MsOH

Large scale synthesis of10-acetyl-N,N,N′N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT)

Acetonitrile (MeCN) (300 l) was added to reactor 1 (R1) and cooled to−5-0° C. Methylthioninium chloride rehydrate (MTC.3H₂O) (150 kg) wasadded and the temperature increased to 15-25° C. Triethylamine (Et₃N)(100 l) was added followed by MeCN rinse (20 l). Hydrazine hydrate(N₂H₄.H₂O) (12 l) was added over 30 min. The reaction temperature wasincreased to 60-70° C. over 1 h and then maintained at this temperaturefor 1 h before being reduced to 40-50° C. Acetic anhydride (Ac₂O) (240l) was added over 1 h followed by MeCN (20 l) rinse. Batch temperaturewas increased to 90-100° C. for 2 h. Temperature was reduced to 55-65°C. and water (340 l) was added over 2 h whilst maintaining thetemperature. The batch temperature was then reduced to −5-5° C. over 2h. and held there for 6 h. The solid was collected by filtration. Thecake was fully de-liquored before water (400 l) was added to R1. Thetemperature in R1 was allowed to rise to 15-25° C. before the water wasused in portions to wash the filter cake. The product was dried under astream of nitrogen for 6 h. before being offloaded (Yield: 90-110 kg).

Large scale purification of10-acetyl-N,N,N′N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT)

Water (300 l) was added to R1, followed by10-Acetyl-N,N,N′N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) (100kg). Toluene (400 l) and 80% aqueous acetic acid (40 l) were added,followed by a water rinse (50 l). The batch temperature was increased to75-85° C. for 1 h. The agitator was stopped and the layers allowed tosettle for 30 min. The lower aqueous layer is removed and fresh water(300 l), 80% aqueous acetic acid (40 l) followed by water rinse (50 l)were then added. The mixture was stirred at 75-85° C. for 1 h before theagitator was stopped and the layers allowed to settle over 30 min. Thelower aqueous layer was removed and fresh water (300 l), 80% aqueousacetic acid (40 l) followed by water rinse (50 l) were then added. Themixture was stirred at 75-85° C. for 1 h before the agitator wasstopped. The layers were allowed to settle for 30 min before the lowerlayer was removed and water (390 l) was added and the mixture stirredfor 1 h. The agitator was stopped and the layers allowed to settle for30 min. The lower aqueous layer was removed and the temperature reducedto −5-5° C. The jacket temperature was increased to 80° C. and then whenit reached 60° C. the temperature was reduced to −10-0° C. over 2 h. Themixture was stirred for 4 h before it was transferred to the filter. Thecake was fully de-liquored before toluene (150 l) was added to R1. Thetoluene was stirred in R1 for 30 min. before it was used in portions towash the filter cake. The product was dried on the filter under a streamof nitrogen for 48 h until loss on drying <1% before being offloaded(Yield: 75-90 kg).

Large scale synthesis ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumbis(methanesulphonate) (LMT.2MsOH)

AcMT (18-22 kg) was added to R1. Toluene (volume (I)=16×AcMT weight) wasadded and the mixture heated to 90-100° C. for 30 min. The solution wasallowed to cool to 60-80° C. before being passed through an in-line 5μfilter to reactor 2 (R2). Toluene (50 l) was added to reactor 1 (stillat ˜70° C.) and stirred for 30 min. This was used to rinse the transferline and filter. The above process was repeated once more. The processof removing the excess toluene from R2 by distillation under reducedpressure was then started. The capacity of R2 permitting, two moreportions of AcMT (18-22 kg each) were transferred from R1 to R2following the method described above. The distillation was complete whenthe batch volume in R2 was reduced to ˜340 I. The temperature wasincreased to 95-105° C. for 15-30 min. before being cooled to 15-25° C.Water (20 l) was added to R2. This was followed by the addition ofmethanesulphonic acid (MSA) (33 I, 99%, 2.2 equiv.) whilst keeping thebatch temperature at 15-30° C. A second portion of water (10 l) wasadded and the mixture stirred at this temperature for 2 h. The mixturewas heated to 80-90° C. for 3-4 h. The biphasic solution was allowed tocool to 48-58° C. before absolute EtOH (75 l) was added over 15-30 min.The stirrer was stopped and the mixture was seeded using 150 g ofcrushed (<100μ) N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumbis(methanesulphonate). A second portion of EtOH (300 l) was added over80-110 min. Jacket temperature was set to 10° C. and when thetemperature reached 25° C. the jacket temperature was reset to 20° C. Itwas stirred at 15-25° C. for 2 h. before the solid was collected byfiltration. The cake was thoroughly de-liquored. MeCN (300 l) was addedto R2 and stirred for 15 min before being used portion-wise to wash thefilter cake. A second 300 I of MeCN was added to R2 and the wash processrepeated. The product was dried on the filter until loss on drying <0.2%before being offloaded (80-90% yield).

Synthesis 2 Synthesis and analysis ofN,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminiumbis(ethanesulfonate) (LMT.2EsOH)

Synthetic Method for LMT.2EsOH

The synthesis of LMT.2EsOH was carried out by acid hydrolysis of10-Acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine. The acidused was ethanesulfonic acid and the solvent combination was aqueousmethanol.

Experimental Details

To a 100 ml round bottom flask was added10-Acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine (5 g,15.27 mmol, MW 327.45 g/mol), (70%, aq) ethanesulfonic acid (7.21 g,45.81 mmol, MW 110.13 g/mol) and methanol (25 ml). The mixture washeated to 75° C. and stirred at this temperature 4 hours before themixture was cooled over ice water. No solid formed and the methanol wasremoved under vacuum to give a viscous green oil. To this oil was addedisopropanol (25 ml) and the mixture was heated to reflux to ensure ahomogenous solution. Once cooled acetone was added until a precipitateformed. The suspension was cooled over ice water for 1 hour before beingfiltered to give the crude product as a yellow solid, which turned greenupon exposure to air. The crude was washed with acetone (3×5 ml) and airdried for 3 days to give the crude product (3.35 g, 43%, MW 505.68g/mol) as a light green solid.

μ_(max) (KBr)/cm⁻¹; 3448 (NH), 3263 (═CH), 3030 (═CH), 2987 (CH), 2938(CH), 2582 (SO₃H), 2452 (SO₃H), 1487 (C—C), 1211 (O═S═O), 1188 (O═S═O),1145 (O═S═O), 1026.

δ_(H)(400 MHz; D₂O): 1.07 (6H, t, J 7.6, CH₃), 2.72 (4H, q, J 7.6,SCH₂), 3.02 (12H, s, N CH₃), 6.54 (2H, d, J 9.2, ArH), 7.02 (4H, brd s,ArH);

δ_(c)(100 MHz; D₂O): 142.3 (QC), 136.6 (QC), 119.9 (CH), 118.4 (QC),118.2 (CH), 115.2 (CH), 46.2 (NCH₃), 45.3 (SCH₂), 8.3 (CH₃).

MP: 208-210° C. (IPA/Acetone)

m/z (EI+): Calculated mass 285.129970; Observed 285.129761 (100%,[M−2EsOH]⁺).

m/z (ES−): Calculated mass 109; Observed 109 (100%, [M−LMT]⁻).

Crystallography

A 1 g sample of LMT.2EsOH was dissolved in acetic acid (˜0.1 g) andethyl acetate was layered on top and allowed to slowly diffuse over 3days in the dark. Crystals developed and were collected and analysed byX-ray diffraction and confirmed the product as the bis(ethanesulfonate).See FIG. 17 a.

Synthesis 3 Synthesis and analysis ofN,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminiumbis(p-toluenesulfonate) (LMT.2TsOH)

Synthetic Method for LMT.2TsOH

The synthesis of LMT.2TsOH was carried out by neutralisingN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium dichloride withsodium carbonate and extracting the neutral species into organicsolvent. The extract was treated with p-toluenesulphonic acid and themixture concentrated to dryness.

Experimental Details

To a 50 ml beaker was added sodium carbonate (0.59 g, 5.58 mmol, MW105.99 g/mol) and water (10 ml), the mixture was stirred until the solidhad dissolved. To a 100 ml separating funnel was addedN,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminium dichloride (1 g,2.79 mmol, MW 358.33 g/mol), tetrahydrofuran (35 ml) and diethylether (5ml) then the aqueous solution of sodium carbonate. The neutral specieswas extracted into the organic solvent layer and separated from theaqueous layer. To the organic extract was added p-toluenesulphonic acidmonohydrate (1.06 g, 5.58 mmol, MW 190.20 g/mol) pre-dissolved intetrahydrofuran (5 ml) and the mixture was concentrated to dryness togive the product (MW 629.8216 g/mol) as a crunchy green amorphous foam.

μ_(max) (KBr)/cm⁻¹; 3440 (NH), 3270 (═CH), 3032 (═CH), 2628 (SO₃, H),1484 (C—C), 1194 (O═S═O), 1122 (O═S═O), 1032.

δ_(H) (400 MHz; D₂O); 2.24 (6H, s, CH₃), 3.09 (12H, s, NCH₃), 6.62 (2H,d, J 8.4, ArH), 7.10 (4H, s, ArH), 7.13 (4H, d, J 8.4, Ts-H), 7.61 (4H,d, J 8.4, Ts-H)

δ_(c) (100 MHz; D₂O); 19.9 (CH₃), 45.9 (NCH₃), 115.0 (CH), 118.2 (CH),118.6 (QC), 119.9 (CH), 125.5 (CH), 128.5 (CH), 137.0 (QC), 140.5 (QC),141.9 (QC), 142.8 (QC).

Mp: 108° C. (THF/Et₂O)

m/z (EI+): Calculated mass 285.129970; Observed 285.129398 (100%,[M−2TsOH]⁺).

m/z (ES−): Calculated mass 171.0116; Observed 171.0121 (100%, [M-LMT]⁻).

Synthesis 4 Synthesis and analysis ofN,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminium ethanedisulfonate(LMT.EDSA)

The synthesis of LMT.EDSA was carried out by acid hydrolysis of10-acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine. The acidused was 1,2-ethanedisulfonic acid and the solvent combination wasaqueous ethanol.

Experimental Details

To a 25 ml round bottom flask was added10-acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine (1 g, 3.05mmol, MW 327.45 g/mol), 1,2-ethanedisulfonic acid monohydrate (0.95 g,4.58 mmol, MW 208.21 g/mol), water (1 ml) and ethanol (5 ml). Themixture was heated to 85° C. and stirred at this temperature for 2.5hours where a yellow green solid precipitated from solution. The slurrywas cooled over ice water for 30 min before filtering to give the crudeproduct as a green yellow solid. The crude was washed with ethanol (3×3ml) and air dried for 15 min before being oven dried for 3.5 hours at70° C. to give the crude product (1.33 g, 91%, MW 475.61 g/mol) as ayellow solid.

Purification of LMT.EDSA

To a 50 ml conical flask was added crude LMT.EDSA (1 g, 2.10 mmol, MW475.61 g/mol) and water (10 ml). The slurry was heated to 95° C. andstirred at this temperature until the solid dissolved. The solution wasthen allowed to cool to 25° C. where a light green crystalline solidformed. The slurry was then cooled over ice water for 30 min beforefiltering. The solid collected was washed with methanol (3×3 ml) and airdried for 18 hours to give the purified product (0.88 g, 88%, MW 475.61g/mol) as a crystalline light green solid.

μ_(max) (KBr)/cm⁻¹; 3408 (NH), 3280 (═CH), 3221 (C—H), 3036 (═CH), 2574(SO₃H), 2480 (SO₃H), 1484 (C—C), 1226 (O═S═O)

δ_(H) (400 MHz; D₂O); 2.98 (12H, s, NCH₃), 3.06 (4H, s, SCH₂), 6.45 (2H,d, J 6, ArH), 6.95 (4H, d J 4, ArH)

δ_(c) (100 MHz; D₂O); 46.2 (NCH₃), 46.4 (SCH₂), 115.1 (CH), 118.1 (CH),118.4 (QC), 119.8 (CH), 136.5 (QC), 142.1 (QC)

MP: decomposes at 268° C. (H₂O)

m/z (EI+): Calculated 285.129970; Observed 285.130948 (100%, [M−EDSA]⁺).

m/z (ES−): Calculated 188.9528; Observed 188.9535 (100%, [M-LMT]⁻).

Crystallography

A 40 mg sample of LMT.EDSA was dissolved in hot deuterated water (˜1 ml)and allowed to slowly cool in the dark. Crystals developed which werecollected and analysed by X-ray diffraction and confirmed the product asthe monohydrate of the 1:1 LMT to EDSA adduct. See FIG. 17 b.

Synthesis 5 Synthesis and analysis ofN,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminiumnaphthalenedisulfonate (LMT.NDSA)

Synthetic Method for LMT.NDSA

The synthesis of LMT.NDSA was carried out by acid hydrolysis of10-acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine. The acidused was 1,5-naphthalenedisulfonic acid and the solvent combination wasaqueous ethanol.

Experimental Details

To a 25 ml round bottom flask was added10-Acetyl-N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine (1 g, 3.05mmol, MW 327.45 g/mol), 1,5-naphthalenedisulfonic acid tetrahydrate(1.65 g, 4.58 mmol, MW 360.36 g/mol), water (1 ml) and ethanol (5 ml).The mixture was heated to 85° C. and stirred at this temperature for 30minutes where the mixture was still insoluble. To the hot mixture wasadded water (4 ml) and the reaction heated to 95° C. and stirred at thistemperature for 8 hours. The suspension was cooled over ice water for 10minutes before being filtered to give the crude product as a light greensolid. The crude was washed with ethanol (3×5 ml) and air dried for 3days to give the crude product (1.75 g, 100%, MW 573.71 g/mol) as alight green blue solid.

μ_(max) (KBr)/cm⁻¹; 3382 (NH), 3302 (═CH), 3040 (═CH), 2525 (SO₃H), 1478(C—C), 1238 (O═S═O), 1219 (O═S═O), 1179, 1158, 1030.

δ_(H) (400 MHz; D₂O); 3.06 (12H, s, NCH₃), 6.70 (2H, brd, ArH), 7.14(4H, brd, ArH), 7.43 (2H, t, J 8.0, 7.6, Naph-H), 7.94 (2H, d, J 7.2,Naph-H), 8.87 (2H, d, J 78.4, Naph-H), 9.10 (1H, s, NH)

δ_(c) (100 MHz; D₂O); 46.0 (NCH₃), 115.3 (CH), 117.5 (QC), 118.7 (CH),120.4 (CH), 124.6 (CH), 124.7 (CH), 129.6 (CH), 129.9 (QC), 138.3 (QC),141.7 (QC), 143.8 (QC).

MP; decomposes at 256° C. (MeCN)

m/z (EI+): Calculated mass 285.129970; Observed 285.130367 (100%,[M-NDSA]⁺).

m/z (ES−): Calculated mass 286.9684; Observed 286.9697 (100%, [M-LMT]⁻).

Example 2 Solubility Studies

i) Solubility of N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumdibromide, dichloride and bis(methanesulphonate) (LMT.2HBr, LMT.2HCl andLMT.2MsOH) salts

Two aqueous solutions (pH 2.00 and 3.01 at 21.4° C.) were prepared bycarefully adding HCl (5 M) to deionised water.

In each experiment a 5 ml aliquot of one of the aforementioned solutionswas heated to 37° C. A portion of the appropriate salt (LMT.2MsOH,LMT.2HCl or LMT.2HBr) was added and the mixture stirred for a fewmoments to allow for complete dissolution of the solid. This step wasrepeated until no further dissolution took place.

The results are shown in the Table:

Salt pH (21.4° C.) g/5 ml* (37° C.) LMT·2HBr 3.01 4.726-5.236 LMT·2HBr2.00 4.822-5.096 LMT·2HCl 3.01 4.978-6.029 LMT·2HCl 2.00 4.404-4.961LMT·2MsOH 2.00 8.825-9.943 *Lower limit of range corresponds to totalweight at which complete dissolution was observed. Upper limit is totalweight added before saturation was achieved

As can be seen LMT.2MsOH has a good aqueous solubility.

ii) pH Dependence of LMT. 2MsOH Salt

In related experiments three buffered stock solutions were prepared (pH2, pH 3, and pH 7) as follows:

pH 2 Buffered Aqueous Solution

A solution of (0.2M) potassium chloride (KCl) (0.745 g in 50 mL ofdeionised water) was initially prepared. From this solution 50 mL wastaken and diluted with approximately 80 mL of deionised water. A (0.2 M)hydrochloric acid (HCl) solution was then used to adjust the pH to 2,before further dilution with deionised water to make up to 200 mL. Afinal pH of 2.00 at 21.6° C. was recorded.

pH 3 Buffered Aqueous Solution

A solution of (0.1M) potassium hydrogen phthalate (2.042 g in 100 mL ofdeionised water) was initially prepared. From this solution 100 mL wastaken and diluted with approximately 50 mL of deionised water. A 0.2 MHCl solution was then used to adjust the pH to 3, before furtherdilution with deionised water to make up to 200 mL. A final pH of 2.99at 21.7° C. was recorded.

pH 7 Buffered Aqueous Solution

A solution of (0.1M) potassium phosphate monobasic (KH₂PO₄) (1.370 g in100 mL of deionised water) was initially prepared. From this solution100 mL was taken and diluted with approximately 80 mL of deionisedwater. A 0.5 M sodium hydroxide (NaOH) solution was then used to adjustthe pH to 7, before further dilution with deionised water to make up to200 mL. A final pH of 7.07 at 22° C. was recorded.

Method

A 5 mL aliquot of an aqueous buffered solution was added to a vial whichcontained a micro-flea. This vial was placed into a water bath set at25° C. To the solution was added LMT.2MsOH in 1-1.5 g portions. Aftereach addition, a 10 minutes stir time was allowed to ensure maximumopportunity for dissolution. The homogeneity of the mixture was judgedby eye. If solid was still present, after the stir time, as judged byvisual inspection, the saturation point was judged to have been reached.

Results

The viscosity of the resulting mixtures precluded adequate isolation ofthe excess solid, therefore it was not possible to determine exactsolubility values. Consequently each of the results are reported as arange in which the total mass of LMT.2MsOH added prior to the saturationpoint constitutes the lower limit and the total mass of LMT.2MsOH added,post saturation point, provides the upper limit.

The results from each of the three experiments are shown below:

pH Solubility (g/mL) 2.00 1.600-1.773 2.99 1.981-2.092 7.07 2.033-2.114

As can be seen, the solubility tailed off slightly as the pH wasreduced, however LMT.2MsOH performed well in each of the three aqueoussystems.

In conclusion LMT.2MsOH has better aqueous solubility than MTC (notshown) and enhanced solubility compared to the corresponding chlorideand bromide salts. This suggests an increased utility in respect of thetreatment and uses described herein.

Example 3 Inhibition of Aggregation and Toxicity

Methods: Solid Phase Assay for Tau Aggregation

The tau-tau aggregation assay uses purified recombinant tau fragments ina solid-phase immunoassay. Methods are described in detail in e.g. WO96/30766. Briefly, the assay measures the binding of truncated tau(amino acids 297-391) in solution to solid-phase bound truncated tau(residues 297-390). Binding of the former is detected with the antibodymAb 423, which specifically recognises peptides containing a C-terminalGlu-391 residue. The Tau complex formed in vitro is similar to theaggregated complex that forms in Alzheimer's disease as a consequence ofthe stability of the pathological Tau-Tau binding interaction through a94/95-amino acid repeat domain (residues 297-390), found in theproteolytically stable core of the paired helical filament.

The B₅₀ value (expressed as mean±SE) is determined as the concentrationof compound at which tau-tau binding is decreased by 50%.

Methods: Cell-Based Tau Aggregation Assay

The assay is based on 3T6 mouse cells that have been engineered toexpress both full-length human tau protein (htau40) under the control ofan inducible promoter (pOPRSVI), and to express low levels of truncatedtau (295-390, dGA) under the control of a constitutive promoter(pcDNA3.1). Expression of large quantities of htau40 is induced by theaddition of IPTG (10-50 μM), which in turn leads to the production ofadditional truncated tau by a process in which aggregation andprocessing of the full length-tau occurs in the presence of dGA tauwhich acts as template. Addition of tau-tau aggregation inhibitors tothe assay blocks this process. Methods are described in more detail inWO 02/055720.

Results are expressed as the concentration at which there is a 50%inhibition of generation of the 12 kD fragment. This is referred to asthe EC₅₀ value.

Cells (4A and clones thereof) are grown to ˜80% confluency in a 10-cmdish, before splitting to two 24-well plates and allowed to grow for 24hrs. Test item is added at various concentrations and, after 24 hrs,IPTG is added. After overnight incubation the medium is removed, thewells are washed with PBS and cells are collected by the addition ofLaemmli buffer. Samples were stored at −20° C. for subsequent gelelectrophoresis, Western blotting and antibody labelling. Samples areseparated by SDS PAGE, transferred to PVDF membrane and the tau labelledwith 7/51 antibody detected by ECL on a Kodak Image Station. Compoundwas typically tested at four concentrations in triplicate over a rangeof concentrations with all the samples being run on one gel. The ratioof the intensities of the dGA to htau40 bands, normalised to controlsamples in which there had been no drug, was plotted against drugconcentration and the EC₅₀ value was determined graphically from theconcentration at which the ratio falls to 0.5.

The method is summarised in Table 1 immediately below. MTC (TR×0014.047)was run as a control in all experiments and the EC₅₀ value wasnormalised to MTC having an EC₅₀=0.59 μM.

TABLE 1 summary of assay procedure for measuring EC₅₀ Timing Action Day1 Split cells to 24-well plates Day 2 Add drug at various concentrationsDay 3 Late afternoon, add IPTG Day 4 Morning, collect in Laemmli buffer,store −20° C. before further processing Processing Day Run samples onSDS Page gels, transfer to PVDF membrane, labeled with 7/51 anti tauantibody. Blots are quantified using the Kodak 1D software and data istransferred to the Systat statistics package for graphing.Methods: Cellular Toxicity Assay

Cells (3T6 mouse fibroblast) are grown to ˜80% confluency in a 10-cmdish, before splitting to 96 well plates, 10% of the 10-cm dish per96-well plate, 50 μl per well. One column of 8 wells is left empty (tobe a reagent blank in the assay). The cells were allowed to growovernight before drug was added to four wells at the startingconcentration (typically 200 μM for MTC or LMT.2HBr) and in subsequentwells using a 1:2 dilution series with the final four wells of cellsbeing used as a control without drug. This allows two drugs to be testedper 96-well plate. The cells were left in the presence of drug for 48hrs, after which medium was removed and cells washed with PBS. Cellnumber was determined using a Cytotox 96 well kit (Promega) which isbased on the lactate dehydrogenase (LDH) assay. The assay quantitativelymeasures LDH, a stable cytosolic enzyme released on cell lysis. ReleasedLDH is measured with an enzymatic assay which results in the conversionof a tetrazolium salt into a red formazan product. The amount of colourformed is proportional to the number of cells lysed.

Briefly, cells are lysed with 50 μl/well 1× lysis buffer for 45-60minutes, followed by 50 μl/well LDH assay reagent for 30 minutes and thereaction stopped with 50 μl/well stop buffer. The absorbance is read at490 nm. The absorbance relative to untreated wells (untreated cells=1.0)was plotted against drug concentration. The LD₅₀ was determinedgraphically from the concentration at which the absorbance is decreasedby 50%. MTC (TR×0014.047) was run as a control in all experiments whentesting LMT.2HBr and the LD₅₀ value was corrected to MTC with an LD₅₀=65μM.

Results:

Various bis(sulfonate) salts according to the invention were tested andcompared with bis(halide) saltsN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium dichloride (LMTC,LMT.2HCl) and N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminiumdi(bromide) LMT.2HBr and with methylthioninium chloride (MTC).

In vitro data for the different methylthioninium salt forms aresummarised in Table 2 immediately below:

TABLE 2 Summary of the in vitro data. Compound LD₅₀ (μM) EC₅₀ (μM) THxB₅₀ (μM) MTC 65 ± 5 0.59 ± 0.04 110 195.6 ± 16.1 (n = 10) LMT·2HBr 61 ±4 (n = 20) 0.66 ± 0.15 (n = 8) 92 472.4 ± 27.6 (n = 3) LMT·2MsOH 34 ± 4(n = 8) 0.19 ± 0.04 (n = 8) 179 238.2 ± 74.2 (n = 3) LMT·2HCl 64 ± 8 (n= 10) 0.63 ± 0.10 (n = 7) 102 360.8 ± 38.2 (n = 3) LMT·2TsOH 87 ± 10 (n= 8) 0.62 ± 0.34 (n = 2) 140 296.0 ± 37.9 (n = 3) LMT·NDSA 77 ± 15 (n =8) 0.71 ± 0.34 (n = 4) 108 333.7 ± 63.2 (n = 2) LMT·EDSA 78 ± 6 (n = 8)0.68 ± 0.32 (n = 4) 115 399.9 ± 17.6 (n = 2) LMT·2EsOH 52 ± 3 (n = 8)0.52 ± 0.13 (n = 3) 100 297.0 ± 75.1 (n = 3) MSA* — NE (20) — >500 EDSA*— NE (20) — >500 THx, therapeutic index (THx = LD₅₀/EC₅₀) Valuesexpressed as mean ± SE. NE = not effective (at max dose tested) *MSA =methansulfonic acid; EDSA = ethanedisulfonic acidComments

The EC50 values (mean±SE) for LMT.2MsOH and LMT.2HCl are 0.19±0.04 μMand 0.63±0.10 μM, respectively, with corresponding therapeutic indicesof 179 and 102.

The relative potency of compounds in the cell-based model of tau-tauaggregation is LMT.2MsOH>MTC, LMT.2HBr, LMT.2HCl. The therapeutic indexis 63% greater for LMT.2MsOH compared with MTC.

The order of potency in the cell-based assay is MTC,LMT.2MsOH>LMT.2HCl>LMT.2HBr. The B50 values for LMT.2MsOH and LMT.2HClare 238.2±74.2 μM and 360.8±38.2 μM, respectively. The order of relativepotency in the cell-free assay is LMT.2MsOH>MTC, LMT.2HCl, LMT.2HBr.

Example 4 Toxicology, Impurities and Effect on the Hemopoietic System

LMT.2HBr, LMT.2HCl, LMT.2MsOH or MTC were administered daily for 14 daysto female Wistar rats; the doses were 95 mg MT/kg/day from Days 1 to 10and 60 mg MT/kg/day from Days 11 to 14. Clinical signs of raised bodyposture, subdued behaviour and general weakness were seen in all treatedgroups. Treatment-related deaths occurred in the LMT.2HBr— andMTC-treated groups.

Changes in red blood cell parameters were seen in the blood and bonemarrow of all treated groups that were indicative of a regenerativeanaemia. These included: decreased numbers of red blood cells, lowhaemoglobin concentration and increased numbers of reticulocytes inblood and an increase in the numbers of red cell precursors in bonemarrow. This was corroborated histologically by increased levels oferythropoiesis in the spleen.

A decrease in the numbers of neutrophilic granulocytes was seen in thebone marrow of all treated animals though the magnitude of this effectwas considerably greater in the LMT.2HBr-treated group than in the othergroups. This difference was also noted in the severity of theneutropaenia observed in prepared blood smears where there was a markeddepletion of mature neutrophils in LMT.2HBr-treated animals, a modestdecrease with MTC and no decrease in the LMT.2HCl or LMT.2MsOH groups.The results of this study suggest that, in rats at least, LMT.2HBr has ahigher propensity to cause neutrophil depletion than LMT.2HCl, LMT.2MsOHor MTC. Decreased numbers of mature neutrophils and granulocytes werealso observed in the bone marrow at the high dose (45 mg MT/kg/day) in a6-month study of LMT.2HBr in the rat. The decreased neutrophils orneutropaenia observed after LMT.2HBr although reversible would makepatients more susceptible to bacterial infections as their primary roleis in destruction of bacteria. Thus LMT.2MsOH shows improved propertiescompared with LMT.2HBr in rats in terms of both tolerability(dose-related deaths) and in neutrophil response.

Table: Neutrophil response in rats following 14 day oral administrationof different salt forms of LMT. Total neutophils are recorded as apercentage of total white cells (approximately 100 white blood cells(range 100 to 107) were examined from each slide; frequency in thepresence of immature neutrophils was recorded per animal group;dose-related deaths recorded as animal numbers per group of 8 rats.

Early Dose-related Compound Neutrophils neutrophils deaths Vehicle15.50% 0/8 0/8 control LMT·2HBr 3.00%* 8/8 2/8 LMT·2HCl 19.90% 2/8 0/8LMT·2MsOH 18.30% 1/8 0/8 *P < 0.001 compared with control

Although LMT.2HCl and LMT.2MsOH are comparable in the above analysis,there is a distinction in the impurities found in the two salt forms.For LMT.2HCl, the presence of methyl chloride was detected duringsynthesis and trapped within the product in such a way that it wasdifficult to remove entirely. By contrast impurities such as ethyl andmethyl methanesulfonate (EMS, MMS) could be controlled to much lowerlevels in the LMT.2MsOH synthetic process

Studies on the hemopoietic system were performed in rat, monkey andminipig.

The lowest doses at which methemoglobinemia was observed were 15 mgMT/kg/day in rats (MTC and LMT.2HBr) or 30 mg MT/kg/day (LMT.2MsOH), 5.3mg MT/kg/day in primates (MTC), and 10 mg MT/kg/day (LMT.2MsOH andLMT.2HBr) in minipigs.

After the first 28-days of dosing in the 9-month LMT.2MsOH study inminipigs, there are no indications of methemoglobinemia at 3 mgMT/kg/day.

However, as would be expected, as dose levels of MTC, LMT.2HBr orLMT.2MsOH increased, signs of oxidative stress to RBCs emerged in adose-related fashion, evidenced by increasing levels of methemoglobinand ultimately at doses that were not tolerated, Heinz body (aggregatesof denatured, precipitated hemoglobin within red cells) formation.

Example 5 Pharmacokinetics

FIG. 18 shows a comparison of the plasma concentration in pig of the MTmoiety over time following dosing of LMT.2HBr, LMT.2HCl and LMT.2MsOH at(two oral doses, 2 and 15 mg/kg).

As can be seen the C_(max) (at T_(max) of 1 hour) for LMT.2MsOH was morethan 2-fold greater than that for LMT.2HCl or LMT.2HBr. Thus LMT.2MsOHcan provide a more effective exposure to MT than LMT.2HCl or LMT.2HBr.

Example 6 Gastric Irritation Studies

Study (28-day rat with MTC or LMT.2HBr): Incidence and severity ofselected microscopic findings in stomach from terminal animals

Incidence and severity of selected findings in sternum, femur, liver andspleen: terminal kill Male Female 1M 2M 3M 4M 5M 6M 7M 1F 2F 3F 4F 5F 6F7F MTC LMT·2HBr MTC LMT·2HBr Tissue and finding Level (mg/kg/day) 0 5 3090 5 30 90 0 5 30 90 5 30 90 No. examined: 5 0 0 5 0 0 5 5 0 0 5 0 0 5Stomach (non glandular) 1 — — 1 — — — Gastritis 2 — 1 — — — 1 3 — — 1 —1 1 Inflammatory cell infiltration 1 — 1 — — — Key: “—” = finding notpresent, 1 = minimal, 2 = slight, 3 = moderate, 4 = moderately severe, 5= severe

From the above the following can be predicted with 10 per group

Incidence and severity of selected findings in sternum, femur, liver andspleen: terminal kill Male Female 1M 2M 3M 4M 5M 6M 7M 1F 2F 3F 4F 5F 6F7F MTC LMT·2HBr MTC LMT·2HBr Tissue and finding Level (mg/kg/day) 0 5 3090 5 30 90 0 5 30 90 5 30 90 No. examined: 10 0 0 10  0 0 10  10 0 0 10 0 0 10  Stomach (non glandular) 1 — — 2 — — — Gastritis 2 — 2 — — — 2 3— — 2 — 2 2 total 2 4 2 4

Study (28-day rat study with LMT.2MsOH): Incidence and severity ofselected microscopic findings in sternum, liver, spleen and stomach fromterminal animals

Incidence and severity of selected findings in sternum, liver andspleen: terminal kill Males Females 1M 2M 3M 4M 1F 2F 3F 4F Tissue andfinding Level (mg/kg/day) 0 5 30 90 0 5 30 90 No. examined: 10 0 0 10 100 0 10 Grade * — Stomach (non glandular) 1 — — — — Gastritis 2 — 2 — 2 3— — — 1 total 2 3 Inflammatory cell infiltration 1 — 4 — 4 2 — — — 1 *Key: “—” = finding not present, 1 = minimal, 2 = slight, 3 = moderate, 4= moderately severe, 5 = severe

These results show that LMT.2MsOH causes less gastric irritation thatLMT.2HBr.

Example 7 Formulations Formulation Example 1 Preparation of LMTM TabletsUsing Direct Compression

Tablets having the following compositions were prepared by a directcompression method:

Tablet strength (LMT mg/tablet) 50 75 100 125 150 Ingredient (mg/tablet)LMTM 84.43 126.65 168.86 211.08 253.29 Spray-dried mannitol 344.57302.35 290.14 392.92 425.71 Microcrystalline 50.00 75.00 95.00 125.00150.00 cellulose (Avicel PH102 or PH112) Crospovidone 15.00 15.00 15.0015.00 15.00 (crosslinked polyvinylpyrrolidone) Magnesium stearate 6.006.00 6.00 6.00 6.00 Total tablet core weight 500.00 525.00 575.00 750.00850.00

The LMTM, spray-dried mannitol, microcrystalline cellulose, crospovidoneand magnesium stearate were blended in a tumbling blender, and thencompressed using a tabletting machine.

The tablet cores were then film coated with an aqueous suspension ofOpadry* blue (*registered trademark of Colorcon for a range of filmcoating materials).

Formulation Example 2 Preparation of LMTM Tablets Using Dry Granulation(Roller Compaction)

Tablets having the following compositions were prepared by a drygranulation method:

Tablet strength (LMT mg/tablet) 50 75 100 125 150 Ingredient (mg/tablet)LMTM 84.43 126.65 168.86 211.08 253.29 Spray-dried mannitol 344.57302.35 290.14 392.92 425.71 Microcrystalline 50.00 75.00 95.00 125.00150.00 cellulose (Avicel PH102 or PH112) Crospovidone 15.00 15.00 15.0015.00 15.00 (crosslinked polyvinylpyrrolidone) Magnesium stearate 6.006.00 6.00 6.00 6.00 Total tablet core weight 500.00 525.00 575.00 750.00850.00

The LMTM, spray-dried mannitol, microcrystalline cellulose, crospovidoneand magnesium stearate were blended in a tumbling blender. The mix wasthen dry granulated using a roller compactor and then milled with anoscillating granulator using a suitable screen. In this case, half ofmagnesium stearate was used prior to roller compaction and half of themagnesium stearate was then added to the granulation and blended priorto compression on a conventional tabletting machine.

The tablet cores were then film coated with an aqueous suspension ofOpadry* blue (*registered trademark of Colorcon for a range of filmcoating materials).

Formulation Example 3 Preparation of LMTM Tablets by Dry Granulation(Slugging)

Tablets having the following compositions were prepared by a further drygranulation method.

Tablet strength (LMT mg/tablet) 50 75 100 125 150 Ingredient (mg/tablet)LMTM 84.43 126.65 168.86 211.08 253.29 Spray-dried mannitol 344.57302.35 290.14 392.92 425.71 Microcrystalline 50.00 75.00 95.00 125.00150.00 cellulose (Avicel PH102 or PH112) Crospovidone 15.00 15.00 15.0015.00 15.00 (crosslinked polyvinylpyrrolidone) Magnesium stearate 6.006.00 6.00 6.00 6.00 Total tablet core weight 500.00 525.00 575.00 750.00850.00

The LMTM and excipients were blended in a tumbling blender, and thencompressed to produce slugs (plain, flat faced tablets) using atabletting machine.

The slugs were then milled using an oscillating granulator fitted with a20 mesh screen.

In this example, half of magnesium stearate was used prior to sluggingand then half of the magnesium stearate added to the granulation andblended prior to compression on a conventional tabletting machine.

The tablet cores were then film coated with an aqueous suspension ofOpadry* blue (*registered trademark of Colorcon for a range of filmcoating materials).

Formulation Example 4 Preparation of LMTM Tablets by Wet Granulation ofExcipients and Incorporation of LMTM Extra-Granularly

Tablets having the following compositions were prepared by a wetgranulation method:

Tablet strength (LMT mg/tablet) 50 75 100 125 150 Ingredient (mg/tablet)LMTM 84.43 126.65 168.86 211.08 253.29 Mannitol 334.57 292.35 280.14380.92 413.71 Microcrystalline 50.00 75.00 95.00 125.00 150.00 cellulose(Avicel PH102) Crospovidone 15.00 15.00 15.00 15.00 15.00 (crosslinkedpolyvinylpyrrolidone) Polyvinylpyrrolidone 10.00 10.00 10.00 12.00 12.00Magnesium stearate 6.00 6.00 6.00 6.00 6.00 Total tablet core weight500.00 525.00 575.00 750.00 850.00

The mannitol, crospovidone (a third of the total) and microcrystallinecellulose were blended in a tumbling blender. The blended material wasthen granulated using a solution of PVP in water. The wet mass was driedin a fluid bed dryer and then milled using an oscillating granulatorfitted with a suitable screen.

The milled material was then blended with the remainder of thecrospovidone and magnesium stearate, and the LMTM, prior to compressionon a conventional tablet machine. The tablet cores were then film coatedwith an aqueous suspension of Opadry* blue (*registered trademark ofColorcon for a range of film coating materials).

Formulation Example 5 Preparation of LMTM Capsules

Capsule strength (LMT mg/capsule) 50 75 100 125 150 200 Ingredientmg/capsule LMTM 84.43 126.65 168.86 211.08 253.29 337.72 Spray-driedmannitol 191.07 148.85 116.64 79.42 42.21 37.78 Crospovidone(crosslinked 3.00 3.00 3.00 3.00 3.00 3.00 polyvinylpyrrolidone)Magnesium stearate 1.50 1.50 1.50 1.50 1.50 1.50 Total capsule fillweight 280.00 280.00 290.00 295.00 300.00 380.00

The LMTM and the excipients were blended in a tumbling blender. Theresulting drug blends were filled into capsules (50, 75, 100, 125 and150 mg formulations into size 1 capsules and the 200 mg formulation intosize 0 capsules) using a capsule filling machine. Both gelatine capsulesand HPMC capsules were prepared.

Formulation Example 6 Results of Stability Testing LMTM 75 mg FilmCoated Tablets

Time Point Storage Location Test (months) 25° C./60% RH 40° C./75% RHAssay as % LMT 0 102.2 102.2 free base 1 101.5 94.8 3 100.0 94.2 6 96.4not done 9 95.6 not done 12 96.0 not done

Formulation Example 7 Results of Stability Testing LMTM 100 mg FilmCoated Tablets

Time Point Storage Location Test (months) 25° C./60% RH 40° C./75% RHAssay as % LMT 0 101.0 101.0 free base 1 96.7 93.7 3 95.9 92.8 6 96.094.2 9 97.1 not done 12 96.8 not done

Formulation Example 8 Results of Stability Testing LMTM 75 mg FilmCoated Tablets

Time Point Storage Location Test (months) 25° C./60% RH 40° C./75% RH %MT formed 0 2.16 2.06 1 2.05 3.79 3 2.19 4.51 6 2.83 5.71 9 3.53 notdone 12 3.28 not done

Formulation Example 9 Results of Stability Testing LMTM 100 mg FilmCoated Tablets

Time Point Storage Location Test (months) 25° C./60% RH 40° C./75% RH %MT formed 0 2.07 2.07 1 1.78 3.27 3 1.81 4.92 6 2.51 5.07 9 2.72 12 2.88

Formulation Example 10

mg/tablet mg/tablet % Material (as LMT) (as LMTM) (core only) TabletCore LMTB 100.00 163.03 32.61 (batch number 0802100070) Spray DriedMannitol 329.00 265.97 53.19 (Pearlitol 200 SD) Microcrystalline 50.0050.00 10.00 cellulose Crospovidone 15.00 15.00 3.00 Magnesium Stearate6.00 6.00 1.20 Tablet Core Total 500.00 500.00 100.00 Film CoatPolyvinyl Alcohol 8.80 8.80 (part hydrolysed) Talc 4.00 4.00 TitaniumDioxide 3.10 3.10 Macrogol PEG 3350 2.47 2.47 Lecithin (soya) 0.70 0.70Iron Oxide Yellow 0.47 0.47 Indigo Carmine 0.45 0.45 Aluminium LakeTotal Film Coated 520.00 520.00 Tablet Manufacturer Piramal, Morpeth, UKTablet Core Batch A02581 Number Date of Manufacture 15^(TH) October 2009

Tablets having the above formulation were prepared by a directcompression method as described above and then film coated (seeFormulation Example 1).

Formulation Example 11 LMTM 75 mg Film-Coated Tablets

mg/tablet mg/tablet % Material (theoretical) (actual) (core only) TabletCore LMTM 75.00 126.80 24.15 (batch numbers 800225510 & 80224450) SprayDried Mannitol 354.00 302.20 57.56 (Pearlitol 200 SD) Microcrystalline75.00 75.00 14.29 cellulose Crospovidone 15.00 15.00 2.86 Magnesiumstearate 6.00 6.00 1.14 Tablet core total 525.00 525.00 100.00 Film CoatPolyvinyl Alcohol 13.86 13.86 (part hydrolysed) Talc 6.30 6.30 TitaniumDioxide 4.89 4.89 Macrogol PEG 3350 3.89 3.89 Lecithin (soya) 1.10 1.10Iron Oxide Yellow 0.75 0.75 Indigo Carmine 0.71 0.71 Aluminium LakeTotal Film Coated 556.5 556.5 Tablet Manufacturer Piramal, Morpeth, UKTablet Core Batch A04827 Number Date of Manufacture 5^(th) August 2010

Tablets having the above formulation were prepared by a directcompression method as described above and then film coated (seeFormulation Example 1).

Formulation Example 12 Dissolution Studies

LMTB film-coated tablets (3×100 mg) and LMTM tablets (4×75 mg), preparedas in Formulation Examples 10 and 11, were stirred (see FIG. 19) at apaddle speed of 50 rpm and the dissolution rate was assessed, using astandard pharmacopeial method (USP 34) and the conditions specifiedbelow.

Instrumental Conditions

Parameter Condition Media 0.1M HCl (Degassed with He purging) MediaVolume 1000 ml, 6 vessels Dissolved Oxygen <3.00 ppm Bath Temperature37° C. ± 0.5° C. Paddles Teflon Coated Paddle Speed 50 rpm Pull Volume10 ml - no media replacement Filter HDPE 10 μm Time points 10, 15, 30and 45 min Vessels 6 (protected from light) λ_(max LMT) 255 nm SampleWorking ca 5 μg/ml (as free base) LMT Concentration (μg/ml) StandardWorking ca 5 μg/ml (as free base) LMT Concentration (μg/ml)

(Q=75% at 45 mins. For S1, 6 of 6 tablets not less than 80% dissolutionat 45 minutes).

Results are shown in the following tables.

LMTM (4×75 mg; Batch No: A04827)

Dissolution (% dissolved):

Vessel T = 10 min T = 15 min T = 30 min T = 45 min 1 94 95 97 99 2 90 9194 95 3 94 94 97 97 4 95 94 97 97 5 92 92 94 94 6 93 92 96 97 Mean 93 9396 97

LMTB (3×100 mg; Batch No: A02581)

Dissolution (% dissolved):

Vessel T = 10 min T = 15 min T = 30 min T = 45 min 1 91 95 96 96 2 96100 99 99 3 95 98 98 99 4 93 95 96 96 5 96 98 99 100 6 98 102 102 102Mean 95 98 98 99

Tablets which had been stored for varying periods of time, under normal(25° C./60% RH) or ‘stressed’ conditions (40° C./75% RH), were alsotested using the same method.

Results are shown in the tables below.

LMTM (4×75 mg: Batch No: A04827)—stored at 25° C./60% RH

Dissolution (% dissolved):

Storage T = Time Vessel T = 10 min T = 15 min T = 30 min 45 min 1 month1 97 97 97 99 2 96 98 101 101 3 98 99 102 102 4 95 97 98 100 5 97 98 101101 6 98 98 100 101 Mean 97 98 100 101 3 months 1 91 93 95 97 2 92 95 9696 3 93 94 95 97 4 92 93 96 96 5 93 94 95 96 6 90 91 94 95 Mean 92 93 9596 6 months 1 89 89 90 91 2 91 90 93 94 3 98 97 98 98 4 97 97 99 99 5 9494 96 96 6 88 90 93 93 Mean 93 93 95 95 9 months 1 92 93 92 94 2 90 9495 97 3 86 91 90 93 4 85 91 96 94 5 90 85 94 94 6 92 96 94 96 Mean 89 9293 94

LMTM (4×75 mg; Batch No: A04827)—stored at 40° C./75% RH

Dissolution (% dissolved):

Storage T = Time Vessel T = 10 min T = 15 min T = 30 min 45 min 1 month1 94 95 97 98 2 94 96 96 97 3 94 96 94 96 4 94 95 95 95 5 100 102 103101 6 93 94 96 97 Mean 95 96 97 97 3 months 1 92 93 95 96 2 93 94 95 973 89 91 92 92 4 89 89 89 91 5 93 95 96 97 6 93 95 98 97 Mean 91 93 94 956 months 1 69 84 92 94 2 93 94 97 91 3 64 85 92 94 4 74 89 92 94 5 91 9595 96 6 73 90 93 94 Mean 77 89 94 94

LMTB (3×100 mg; Batch No: A02581)—stored at 25° C./60% RH

Dissolution (% dissolved):

Storage T = T = Time Vessel T = 10 min T = 15 min 30 min 45 min 3 weeks1 96 98 98 98 2 94 97 97 98 3 94 97 97 97 4 98 100 101 101 5 92 94 95 956 92 95 97 97 Mean 94 97 98 98 3 months 1 89 92 92 92 2 89 92 93 92 3 9396 96 96 4 95 98 99 98 5 95 96 96 96 6 96 99 98 97 Mean 93 96 96 95 6months 1 96 97 96 97 2 95 101 100 101 3 95 97 96 97 4 95 95 95 96 5 9698 99 99 6 95 94 94 96 Mean 95 97 97 98 9 months 1 87 91 93 91 2 88 9294 92 3 90 93 91 92 4 91 95 93 94 5 91 93 93 92 6 94 95 95 93 Mean 90 9393 92 12 months 1 92 97 98 97 2 91 92 92 92 3 95 96 95 96 4 94 95 95 955 89 89 89 89 6 97 98 98 98 Mean 93 94 95 94

LMTB (3×100 mg; Batch No: A02581)—stored at 40° C./75% RH

Dissolution (% dissolved):

Storage T = Time Vessel T = 10 min T = 15 min T = 30 min 45 min 3 weeks1 94 98 99 98 2 96 100 100 101 3 94 97 96 97 4 94 98 98 98 5 95 97 98 986 95 97 98 97 Mean 95 98 98 98 3 months 1 92 93 94 93 2 93 97 97 97 3 9092 92 92 4 84 89 94 94 5 84 97 97 97 6 93 94 93 94 Mean 89 94 95 95 6months 1 8 72 96 96 2 48 82 95 96 3 91 93 94 94 4 94 98 98 99 5 13 71 9393 6 74 87 92 93 Mean 55 84 95 95Annex—Crystallographic Data

Crystallographic Data for LMT. EDSA (FIG. 17 a):

TABLE 1 Crystal data and structure refinement for LMT·EDSA.Identification code 6408CM136 Empirical formula C₁₈H₂₇N₃O₇S₃ Formulaweight 493.62 Temperature 100(2) K Wavelength 0.71073 Å Crystal systemMonoclinic Space group C2/c Unit cell dimensions a = 18.2832(3) Å α =90°. b = 11.8667(3) Å β = 114.1990(10)°. c = 10.9539(2) Å γ = 90°.Volume 2167.74(8) Å³ Z 4 Density (calculated) 1.519 Mg/m³ Absorptioncoefficient 0.389 mm⁻¹ F(000) 1048 Crystal size 0.28 × 0.21 × 0.18 mm³Theta range for data collection 2.11 to 27.51°. Index ranges −23 <= h <=23, −15 <= k <= 15, −14 <= l <= 14 Reflections collected 25214Independent reflections 2487 [R(int) = 0.0486] Completeness to theta =25.00° 99.9% Absorption correction Semi-empirical from equivalents Max.and min. transmission 0.9333 and 0.8989 Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 2487/0/144Goodness-of-fit on F² 1.080 Final R indices [I > 2sigma(I)] R1 = 0.0315,wR2 = 0.0906 R indices (all data) R1 = 0.0336, wR2 = 0.0925 Largestdiff. peak and hole 0.333 and −0.654 e · Å⁻³

TABLE 2 Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for LMT·EDSA. U(eq) is defined as one third of thetrace of the orthogonalized U^(ij) tensor. x y z U(eq) S(1) 100002270(1) 12500 19(1) S(2)  3802(1) 204(1)  9934(1) 11(1) N(1) 10000−370(1) 12500 18(1) N(2)  7750(1) 1619(1)  7782(1) 12(1) O(1)  3943(1)−435(1) 11137(1) 18(1) O(2)  3830(1) 1425(1) 10131(1) 17(1) O(3) 3063(1) −144(1)  8793(1) 15(1) C(1)  9411(1) 1332(1) 11218(1) 13(1)C(2)  9493(1) 154(1) 11332(1) 14(1) C(3)  9040(1) −512(1) 10228(1) 16(1)C(4)  8481(1) −30(1)  9061(1) 16(1) C(5)  8383(1) 1126(1)  8996(1) 13(1)C(6)  8852(1) 1814(1) 10051(1) 13(1) C(8)  7127(1) 2225(1)  8087(1)16(1) C(9)  8070(1) 2352(1)  7003(1) 17(1) C(10)  4593(1) −141(1) 9448(1) 13(1) O(1S)  5000 2293(1)  2500 24(1)

TABLE 3 Bond lengths [Å] and angles [°] for LMT·EDSA. S(1)—C(1)1.7696(13) S(1)—C(1)#1 1.7696(13) S(2)—O(1) 1.4488(10) S(2)—O(2)1.4629(10) S(2)—O(3) 1.4747(10) S(2)—C(10) 1.7802(13) N(1)—C(2)1.3826(15) N(1)—C(2)#1 1.3826(15) N(1)—H(1) 0.8800 N(2)—C(5) 1.4785(16)N(2)—C(9) 1.4959(17) N(2)—C(8) 1.4970(17) N(2)—H(2) 0.9300 C(1)—C(6)1.3913(17) C(1)—C(2) 1.4049(18) C(2)—C(3) 1.3972(19) C(3)—C(4)1.3908(19) C(3)—H(3) 0.9500 C(4)—C(5) 1.3804(19) C(4)—H(4) 0.9500C(5)—C(6) 1.3881(18) C(6)—H(6) 0.9500 C(8)—H(8A) 0.9800 C(8)—H(8B)0.9800 C(8)—H(8C) 0.9800 C(9)—H(9A) 0.9800 C(9)—H(9B) 0.9800 C(9)—H(9C)0.9800 C(10)—C(10)#2 1.522(2) C(10)—H(10A) 0.9900 C(10)—H(10B) 0.9900O(1S)—H(1O1) 0.7486 O(1S)—H(2O1) 0.9717 C(1)—S(1)—C(1)#1 102.05(9)O(1)—S(2)—O(2) 113.66(6) O(1)—S(2)—O(3) 112.60(6) O(2)—S(2)—O(3)111.52(6) O(1)—S(2)—C(10) 106.77(6) O(2)—S(2)—C(10) 106.71(6)O(3)—S(2)—C(10) 104.89(6) C(2)—N(1)—C(2)#1 126.50(17) C(2)—N(1)—H(1)116.7 C(2)#1—N(1)—H(1) 116.7 C(5)—N(2)—C(9) 113.51(10) C(5)—N(2)—C(8)112.13(10) C(9)—N(2)—C(8) 111.06(11) C(5)—N(2)—H(2) 106.5 C(9)—N(2)—H(2)106.5 C(8)—N(2)—H(2) 106.5 C(6)—C(1)—C(2) 120.17(12) C(6)—C(1)—S(1)116.69(10) C(2)—C(1)—S(1) 123.14(10) N(1)—C(2)—C(3) 118.76(13)N(1)—C(2)—C(1) 122.44(13) C(3)—C(2)—C(1) 118.79(12) C(4)—C(3)—C(2)120.95(13) C(4)—C(3)—H(3) 119.5 C(2)—C(3)—H(3) 119.5 C(5)—C(4)—C(3)119.15(13) C(5)—C(4)—H(4) 120.4 C(3)—C(4)—H(4) 120.4 C(4)—C(5)—C(6)121.24(12) C(4)—C(5)—N(2) 118.50(12) C(6)—C(5)—N(2) 120.25(12)C(5)—C(6)—C(1) 119.54(12) C(5)—C(6)—H(6) 120.2 C(1)—C(6)—H(6) 120.2N(2)—C(8)—H(8A) 109.5 N(2)—C(8)—H(8B) 109.5 H(8A)—C(8)—H(8B) 109.5N(2)—C(8)—H(8C) 109.5 H(8A)—C(8)—H(8C) 109.5 H(8B)—C(8)—H(8C) 109.5N(2)—C(9)—H(9A) 109.5 N(2)—C(9)—H(9B) 109.5 H(9A)—C(9)—H(9B) 109.5N(2)—C(9)—H(9C) 109.5 H(9A)—C(9)—H(9C) 109.5 H(9B)—C(9)—H(9C) 109.5C(10)#2—C(10)—S(2) 111.21(12) C(10)#2—C(10)—H(10A) 109.4S(2)—C(10)—H(10A) 109.4 C(10)#2—C(10)—H(10B) 109.4 S(2)—C(10)—H(10B)109.4 H(10A)—C(10)—H(10B) 108.0 H(1O1)—O(1S)—H(2O1) 100.8

Symmetry transformations used to generate equivalent atoms:

#1−x+2,y,−z+5/2 #2−x+1,−y,−z+2

TABLE 4 Anisotropic displacement parameters (Å² × 10³) for LMT·EDSA. Theanisotropic displacement factor exponent takes the form: −2p²[h²a*²U¹¹ + . . . + 2 h k a* b* U¹²] U¹¹ U²² U³³ U²³ U¹³ U¹²  S(1) 20(1)10(1) 14(1) 0 −6(1)  0  S(2) 10(1) 12(1)  9(1) 0(1) 2(1)  0(1) N(1)22(1)  9(1) 14(1) 0 −3(1)  0 N(2) 12(1) 13(1)  9(1) 0(1) 2(1) −1(1) O(1)16(1) 22(1) 13(1) 5(1) 5(1)  0(1) O(2) 17(1) 13(1) 17(1) −2(1)  4(1) 0(1) O(3) 11(1) 16(1) 13(1) −1(1)  0(1) −1(1) C(1) 12(1) 13(1) 11(1)−1(1)  2(1) −2(1) C(2) 12(1) 13(1) 13(1) 0(1) 2(1)  0(1) C(3) 19(1)11(1) 14(1) −2(1)  3(1) −1(1) C(4) 16(1) 15(1) 12(1) −2(1)  2(1) −1(1)C(5) 12(1) 15(1) 10(1) 1(1) 2(1)  0(1) C(6) 13(1) 12(1) 12(1) 0(1) 4(1) 0(1) C(8) 13(1) 18(1) 15(1) 1(1) 5(1)  1(1) C(9) 18(1) 22(1) 12(1) 2(1)6(1) −2(1)  C(10) 11(1) 17(1) 10(1) −2(1)  3(1) −1(1)  O(1S) 25(1) 14(1)18(1) 0 −7(1)  0

TABLE 5 Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for LMT·EDSA. x y z U(eq) H(1) 10000 −1111 1250022 H(2) 7492 1019 7227 15 H(3) 9114 −1305 10275 19 H(4) 8171 −489 831919 H(6) 8791 2610 9978 15 H(8A) 6937 1734 8616 23 H(8B) 6675 2426 724823 H(8C) 7359 2911 8596 23 H(9A) 8321 3023 7531 26 H(9B) 7629 2582 616426 H(9C) 8469 1933 6802 26 H(10A) 4571 −955 9239 16 H(10B) 4521 284 862816 H(1O1) 5146 2050 3190 29 H(2O1) 4556 1790 2015 29

Crystallographic Data for LMT. 2EsOH (FIG. 17 b)

TABLE 1 Crystal data and structure refinement for LMT·2EsOH.Identification code 6408cm173c_0m Empirical formula C₂₀H₃₁N₃O₆S₃ Formulaweight 505.66 Temperature 100(2) K Wavelength 0.71073 Å Crystal systemMonoclinic Space group C2/c Unit cell dimensions a = 40.8384(12) Å α =90°. b = 25.2658(7) Å β = 115.4540(10)°. c = 20.3833(6) Å γ = 90°.Volume 18990.2(9) Å³ Z 32 Density (calculated) 1.415 Mg/m³ Absorptioncoefficient 0.354 mm⁻¹ F(000) 8576 Crystal size 0.32 × 0.24 × 0.18 mm³Theta range for data collection 0.98 to 25.00°. Index ranges −48 <= h <=48, −29 <= k <= 30, −24 <= l <= 23 Reflections collected 108984Independent reflections 16707 [R(int) = 0.0912] Completeness to theta =99.9% 25.00° Absorption correction Semi-empirical from equivalents Max.and min. transmission 0.9391 and 0.8952 Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 16707/25/1205Goodness-of-fit on F² 1.085 Final R indices R1 = 0.0628, wR2 = 0.1638[I > 2sigma(I)] R indices (all data) R1 = 0.0986, wR2 = 0.1918 Largestdiff. peak and hole 2.683 and −0.811 e · Å⁻³

TABLE 2 Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for LMT·2EsOH. U(eq) is defined as one third ofthe trace of the orthogonalized U^(ij) tensor. x y z U(eq) S(1A)  256(1)3950(1) 1866(1) 69(1) N(1A) −401(1) 3176(1) 1220(2) 29(1) N(2A)  980(1)2230(1) 2700(2) 25(1) N(3A) −743(1) 5348(1)  744(2) 25(1) C(1A) −207(1)4102(2) 1408(3) 33(1) C(2A) −297(1) 4633(2) 1295(3) 32(1) C(3A) −652(1)4786(2)  916(2) 24(1) C(4A) −926(1) 4413(2)  664(2) 27(1) C(5A) −836(1)3880(2)  784(3) 28(1) C(6A) −479(1) 3714(2) 1142(2) 25(1) C(7A)  −60(1)2948(2) 1584(2) 25(1) C(8A)  −27(1) 2402(2) 1631(2) 24(1) C(9A)  308(1)2154(2) 1988(2) 25(1) C(10A)  614(1) 2468(2) 2318(2) 24(1) C(11A) 588(1) 3011(2) 2281(3) 29(1) C(12A)  254(1) 3255(2) 1917(3) 32(1)C(13A) 1017(1) 1896(2) 3334(3) 36(1) C(14A) 1092(1) 1933(2) 2196(3)41(1) C(15A) −662(2) 5526(2)  130(3) 50(2) C(16A) −570(1) 5712(2)1375(3) 33(1) S(1B) 2816(1) 1091(1) 1548(1) 26(1) N(1B) 2213(1)  258(1)1144(2) 21(1) N(2B) 3639(1) −517(1) 2667(2) 27(1) N(3B) 1743(1) 2378(1) 956(2) 22(1) C(1B) 2355(1) 1198(2) 1329(2) 19(1) C(2B) 2237(1) 1719(2)1291(2) 22(1) C(3B) 1873(1) 1825(2) 1032(2) 21(1) C(4B) 1620(1) 1417(2) 832(2) 22(1) C(5B) 1738(1)  896(2)  890(2) 21(1) C(6B) 2104(1)  778(2)1128(2) 21(1) C(7B) 2566(1)  73(2) 1537(2) 22(1) C(8B) 2628(1) −468(2)1684(2) 23(1) C(9B) 2974(1) −666(2) 2055(2) 24(1) C(10B) 3263(1) −320(2)2304(2) 24(1) C(11B) 3213(1)  220(2) 2178(2) 23(1) C(12B) 2866(1) 417(2) 1788(2) 22(1) C(13B) 3693(2) −973(2) 3183(3) 38(1) C(14B)3785(1) −651(2) 2126(2) 29(1) C(15B) 1835(1) 2684(2)  426(3) 34(1)C(16B) 1872(1) 2660(2) 1668(3) 35(1) S(1C) 5390(1) 3672(1) 1826(1) 25(1)N(1C) 4792(1) 2826(1) 1436(2) 25(1) N(2C) 6224(1) 2099(1) 3029(2) 20(1)N(3C) 4310(1) 4945(1) 1101(2) 26(1) C(1C) 4925(1) 3774(2) 1581(2) 20(1)C(2C) 4803(1) 4289(2) 1510(2) 21(1) C(3C) 4436(1) 4388(2) 1235(2) 21(1)C(4C) 4184(1) 3984(2) 1037(2) 25(1) C(5C) 4307(1) 3464(2) 1126(2) 23(1)C(6C) 4676(1) 3350(2) 1388(2) 21(1) C(7C) 5145(1) 2651(2) 1833(2) 22(1)C(8C) 5213(1) 2113(2) 1990(2) 22(1) C(9C) 5559(1) 1925(2) 2384(2) 21(1)C(10C) 5847(1) 2278(2) 2639(2) 20(1) C(11C) 5788(1) 2813(2) 2493(2)21(1) C(12C) 5443(1) 3002(2) 2087(2) 20(1) C(13C) 6374(1) 1945(2)2499(2) 26(1) C(14C) 6284(1) 1670(2) 3576(2) 24(1) C(15C) 4375(2)5182(2)  496(3) 47(1) C(16C) 4468(2) 5280(2) 1771(3) 47(2) S(1D) 7907(1)1349(1) 2060(1) 32(1) N(1D) 7269(1)  547(1) 1633(2) 29(1) N(2D) 8670(1)−331(2) 2894(2) 28(1) N(3D) 6848(1) 2694(1) 1136(2) 27(1) C(1D) 7440(1)1484(2) 1723(2) 25(1) C(2D) 7333(1) 2011(2) 1602(2) 27(1) C(3D) 6969(1)2136(2) 1304(2) 26(1) C(4D) 6709(1) 1744(2) 1117(3) 32(1) C(5D) 6818(1)1220(2) 1238(3) 32(1) C(6D) 7179(1) 1080(2) 1536(2) 24(1) C(7D) 7614(1) 338(2) 1971(2) 22(1) C(8D) 7660(1) −209(2) 2063(2) 24(1) C(9D) 8001(1)−439(2) 2370(2) 25(1) C(10D) 8302(1) −111(2) 2608(2) 24(1) C(11D)8268(1)  437(2) 2537(2) 26(1) C(12D) 7925(1)  662(2) 2212(2) 23(1)C(13D) 8743(1) −762(2) 3435(3) 43(1) C(14D) 8764(1) −504(2) 2294(3)31(1) C(15D) 6807(2) 2848(2)  397(3) 41(1) C(16D) 7078(1) 3086(2)1700(3) 36(1) S(2)  525(1)  500(1) 1331(1) 35(1) S(3)  827(1) 2850(1) 152(1) 25(1) S(4) 1743(1) 4426(1)  389(1) 29(1) S(5) 3048(1) 3051(1)1414(1) 30(1) S(6) 5663(1)  497(1) 1723(1) 33(1) S(7) 5874(1) 2562(1) 455(1) 34(1) S(8) 8160(1) 2896(1) 1391(1) 23(1) S(9) 8481(1)  615(1) 291(1) 26(1) O(1)  619(4)  884(5) 1948(7) 56(2) O(2)  392(10)   52(10) 1452(19) 70(6) O(3)  886(4)  594(6) 1706(8) 55(2) O(1′)  342(2)  769(3)1689(4) 61(2) O(2′)  419(6)  −77(6)  1226(11) 70(5) O(3′)  893(2) 480(4) 1357(5) 58(2) O(4) 1009(1) 2335(1)  358(2) 35(1) O(5) 1031(1)3269(1)  635(2) 41(1) O(6)  718(1) 2970(1) −613(2) 27(1) O(7) 1911(1)3966(1)  826(2) 45(1) O(8) 1422(1) 4299(1) −282(2) 45(1) O(9) 1667(1)4851(1)  790(2) 30(1) O(10) 3321(1) 2861(1) 1185(2) 41(1) O(11) 2835(1)2637(2) 1525(3) 68(1) O(12) 3217(1) 3392(1) 2047(2) 46(1) O(13) 5318(1) 542(2) 1772(2) 56(1) O(14) 5728(1)  −26(1) 1506(2) 60(1) O(15) 5964(1)678(1) 2388(2) 42(1) O(16) 5932(1) 2520(1) −212(2) 32(1) O(17) 5774(1)2071(2)  681(2) 57(1) O(18) 6182(1) 2836(1) 1038(2) 39(1) O(19) 8126(1)2360(1) 1113(2) 31(1) O(20) 8540(1) 3032(1) 1876(2) 35(1) O(21) 7918(1)3019(1) 1720(2) 31(1) O(22) 8264(1)  640(1) −494(2) 26(1) O(23) 8590(1) 62(1)  534(2) 31(1) O(24) 8786(1)  983(1)  553(2) 33(1) C(1S)  298(2) 782(2)  463(4) 62(2) C(2S)  387(2) 1337(2)  396(3) 45(1) C(3S) 2064(1)4682(2)  95(3) 30(1) C(4S) 1928(1) 5196(2) −337(3) 39(1) C(5S) 2748(1)3452(2)  689(3) 37(1) C(6S) 2934(1) 3925(2)  551(3) 32(1) C(7S) 5504(1)2984(2)  239(3) 39(1) C(8S) 5570(2) 3516(2)  −63(4) 54(2) C(9S) 5626(2) 921(2) 1016(3) 38(1) C(10S) 5990(3)  881(4) 989(5) 100(3)  C(11S)8047(1) 3340(2) 650(3) 28(1) C(12S) 7648(1) 3320(2) 118(3) 38(1) C(13S)8200(1)  808(2)  708(3) 27(1) C(14S) 7876(1)  454(2)  490(3) 34(1)C(15S)  414(1) 2769(2)  254(3) 36(1) C(16S)  186(2) 3261(2)  66(3) 49(2)

TABLE 3 Bond lengths [Å] and angles [°] for LMT·2EsOH. S(1A)—C(1A)1.756(5) S(1A)—C(12A) 1.759(5) N(1A)—C(7A) 1.390(6) N(1A)—C(6A) 1.389(6)N(1A)—H(1AA) 0.8800 N(2A)—C(10A) 1.485(5) N(2A)—C(14A) 1.492(6)N(2A)—C(13A) 1.495(6) N(2A)—H(2AA) 0.9300 N(3A)—C(3A) 1.474(5)N(3A)—C(16A) 1.489(6) N(3A)—C(15A) 1.494(6) N(3A)—H(3A) 0.9300C(1A)—C(2A) 1.384(6) C(1A)—C(6A) 1.404(6) C(2A)—C(3A) 1.371(6)C(2A)—H(2A) 0.9500 C(3A)—C(4A) 1.384(6) C(4A)—C(5A) 1.389(6) C(4A)—H(4A)0.9500 C(5A)—C(6A) 1.384(6) C(5A)—H(5A) 0.9500 C(7A)—C(8A) 1.387(6)C(7A)—C(12A) 1.399(6) C(8A)—C(9A) 1.391(6) C(8A)—H(8A) 0.9500C(9A)—C(10A) 1.384(6) C(9A)—H(9A) 0.9500 C(10A)—C(11A) 1.376(6)C(11A)—C(12A) 1.388(6) C(11A)—H(11A) 0.9500 C(13A)—H(13A) 0.9800C(13A)—H(13B) 0.9800 C(13A)—H(13C) 0.9800 C(14A)—H(14A) 0.9800C(14A)—H(14B) 0.9800 C(14A)—H(14C) 0.9800 C(15A)—H(15A) 0.9800C(15A)—H(15B) 0.9800 C(15A)—H(15C) 0.9800 C(16A)—H(16A) 0.9800C(16A)—H(16B) 0.9800 C(16A)—H(16C) 0.9800 S(1B)—C(12B) 1.761(4)S(1B)—C(1B) 1.762(4) N(1B)—C(6B) 1.385(5) N(1B)—C(7B) 1.392(6)N(1B)—H(1B) 0.8800 N(2B)—C(10B) 1.474(6) N(2B)—C(14B) 1.502(6)N(2B)—C(13B) 1.512(6) N(2B)—H(2BB) 0.9300 N(3B)—C(3B) 1.479(5)N(3B)—C(16B) 1.496(6) N(3B)—C(15B) 1.501(6) N(3B)—H(3B) 0.9300C(1B)—C(2B) 1.392(6) C(1B)—C(6B) 1.410(6) C(2B)—C(3B) 1.372(6)C(2B)—H(2B) 0.9500 C(3B)—C(4B) 1.391(6) C(4B)—C(5B) 1.391(6) C(4B)—H(4B)0.9500 C(5B)—C(6B) 1.389(6) C(5B)—H(5B) 0.9500 C(7B)—C(8B) 1.399(6)C(7B)—C(12B) 1.406(6) C(8B)—C(9B) 1.377(6) C(8B)—H(8B) 0.9500C(9B)—C(10B) 1.381(6) C(9B)—H(9B) 0.9500 C(10B)—C(11B) 1.384(6)C(11B)—C(12B) 1.388(6) C(11B)—H(11B) 0.9500 C(13B)—H(13D) 0.9800C(13B)—H(13E) 0.9800 C(13B)—H(13F) 0.9800 C(14B)—H(14D) 0.9800C(14B)—H(14E) 0.9800 C(14B)—H(14F) 0.9800 C(15B)—H(15D) 0.9800C(15B)—H(15E) 0.9800 C(15B)—H(15F) 0.9800 C(16B)—H(16D) 0.9800C(16B)—H(16E) 0.9800 C(16B)—H(16F) 0.9800 S(1C)—C(12C) 1.760(4)S(1C)—C(1C) 1.765(4) N(1C)—C(7C) 1.387(5) N(1C)—C(6C) 1.395(5)N(1C)—H(1C) 0.8800 N(2C)—C(10C) 1.469(5) N(2C)—C(14C) 1.498(5)N(2C)—C(13C) 1.503(5) N(2C)—H(2CC) 0.9300 N(3C)—C(3C) 1.482(5)N(3C)—C(15C) 1.494(6) N(3C)—C(16C) 1.497(6) N(3C)—H(3C) 0.9300C(1C)—C(2C) 1.377(6) C(1C)—C(6C) 1.411(6) C(2C)—C(3C) 1.380(6)C(2C)—H(2C) 0.9500 C(3C)—C(4C) 1.381(6) C(4C)—C(5C) 1.390(6) C(4C)—H(4C)0.9500 C(5C)—C(6C) 1.397(6) C(5C)—H(5C) 0.9500 C(7C)—C(8C) 1.397(6)C(7C)—C(12C) 1.413(6) C(8C)—C(9C) 1.378(6) C(8C)—H(8C) 0.9500C(9C)—C(10C) 1.386(6) C(9C)—H(9C) 0.9500 C(10C)—C(11C) 1.384(6)C(11C)—C(12C) 1.377(6) C(11C)—H(11C) 0.9500 C(13C)—H(13G) 0.9800C(13C)—H(13H) 0.9800 C(13C)—H(13I) 0.9800 C(14C)—H(14G) 0.9800C(14C)—H(14H) 0.9800 C(14C)—H(14I) 0.9800 C(15C)—H(15G) 0.9800C(15C)—H(15H) 0.9800 C(15C)—H(15I) 0.9800 C(16C)—H(16G) 0.9800C(16C)—H(16H) 0.9800 C(16C)—H(16I) 0.9800 S(1D)—C(12D) 1.760(4)S(1D)—C(1D) 1.761(5) N(1D)—C(7D) 1.381(6) N(1D)—C(6D) 1.389(6)N(1D)—H(1D) 0.8800 N(2D)—C(10D) 1.471(6) N(2D)—C(13D) 1.486(6)N(2D)—C(14D) 1.495(6) N(2D)—H(2D) 0.9300 N(3D)—C(3D) 1.483(6)N(3D)—C(15D) 1.495(6) N(3D)—C(16D) 1.504(6) N(3D)—H(3D) 0.9300C(1D)—C(2D) 1.389(6) C(1D)—C(6D) 1.405(6) C(2D)—C(3D) 1.379(6)C(2D)—H(2DD) 0.9500 C(3D)—C(4D) 1.381(6) C(4D)—C(5D) 1.385(7)C(4D)—H(4D) 0.9500 C(5D)—C(6D) 1.377(6) C(5D)—H(5D) 0.9500 C(7D)—C(8D)1.394(6) C(7D)—C(12D) 1.409(6) C(8D)—C(9D) 1.385(6) C(8D)—H(8D) 0.9500C(9D)—C(10D) 1.384(6) C(9D)—H(9D) 0.9500 C(10D)—C(11D) 1.391(6)C(11D)—C(12D) 1.388(6) C(11D)—H(11D) 0.9500 C(13D)—H(13J) 0.9800C(13D)—H(13K) 0.9800 C(13D)—H(13L) 0.9800 C(14D)—H(14J) 0.9800C(14D)—H(14K) 0.9800 C(14D)—H(14L) 0.9800 C(15D)—H(15J) 0.9800C(15D)—H(15K) 0.9800 C(15D)—H(15L) 0.9800 C(16D)—H(16J) 0.9800C(16D)—H(16K) 0.9800 C(16D)—H(16L) 0.9800 S(2)—O(2) 1.32(3) S(2)—O(3)1.359(16) S(2)—O(1′) 1.423(7) S(2)—O(3′) 1.481(8) S(2)—O(2′) 1.510(16)S(2)—O(1) 1.503(13) S(2)—C(1S) 1.756(6) S(3)—O(5) 1.442(3) S(3)—O(6)1.459(3) S(3)—O(4) 1.469(3) S(3)—C(15S) 1.795(5) S(4)—O(7) 1.446(4)S(4)—O(9) 1.461(3) S(4)—O(8) 1.466(3) S(4)—C(3S) 1.779(5) S(5)—O(11)1.438(4) S(5)—O(12) 1.456(4) S(5)—O(10) 1.463(3) S(5)—C(5S) 1.775(5)S(6)—O(14) 1.453(4) S(6)—O(15) 1.456(4) S(6)—O(13) 1.458(4) S(6)—C(9S)1.749(5) S(7)—O(17) 1.442(4) S(7)—O(18) 1.479(4) S(7)—O(16) 1.482(3)S(7)—C(7S) 1.744(5) S(8)—O(21) 1.447(3) S(8)—O(19) 1.452(3) S(8)—O(20)1.479(3) S(8)—C(11S) 1.776(4) S(9)—O(24) 1.458(3) S(9)—O(22) 1.458(3)S(9)—O(23) 1.486(3) S(9)—C(13S) 1.765(5) O(1)—O(3) 1.56(2) C(1S)—C(2S)1.470(8) C(1S)—H(1S1) 0.9900 C(1S)—H(1S2) 0.9900 C(2S)—H(2S1) 0.9800C(2S)—H(2S2) 0.9800 C(2S)—H(2S3) 0.9800 C(3S)—C(4S) 1.534(7)C(3S)—H(3S1) 0.9900 C(3S)—H(3S2) 0.9900 C(4S)—H(4S1) 0.9800 C(4S)—H(4S2)0.9800 C(4S)—H(4S3) 0.9800 C(5S)—C(6S) 1.506(7) C(5S)—H(5S1) 0.9900C(5S)—H(5S2) 0.9900 C(6S)—H(6S1) 0.9800 C(6S)—H(6S2) 0.9800 C(6S)—H(6S3)0.9800 C(7S)—C(8S) 1.550(7) C(7S)—H(7S1) 0.9900 C(7S)—H(7S2) 0.9900C(8S)—H(8S1) 0.9800 C(8S)—H(8S2) 0.9800 C(8S)—H(8S3) 0.9800 C(9S)—C(10S)1.511(10) C(9S)—H(9S1) 0.9900 C(9S)—H(9S2) 0.9900 C(10S)—11(10A) 0.9800C(10S)—H(10B) 0.9800 C(10S)—H(10C) 0.9800 C(11S)—C(12S) 1.522(7)C(11S)—H(11E) 0.9900 C(11S)—H(11F) 0.9900 C(12S)—H(12A) 0.9800C(12S)—H(12B) 0.9800 C(12S)—H(12C) 0.9800 C(13S)—C(14S) 1.498(6)C(13S)—H(13M) 0.9900 C(13S)—H(13N) 0.9900 C(14S)—H(14M) 0.9800C(14S)—H(14N) 0.9800 C(14S)—H(14O) 0.9800 C(15S)—C(16S) 1.500(7)C(15S)—H(15M) 0.9900 C(15S)—H(15N) 0.9900 C(16S)—H(16M) 0.9800C(16S)—H(16N) 0.9800 C(16S)—H(16O) 0.9800 C(1A)—S(1A)—C(12A) 102.6(2)C(7A)—N(1A)—C(6A) 126.5(4) C(7A)—N(1A)—H(1AA) 116.8 C(6A)—N(1A)—H(1AA)116.8 C(10A)—N(2A)—C(14A) 112.3(4) C(10A)—N(2A)—C(13A) 112.8(4)C(14A)—N(2A)—C(13A) 111.3(4) C(10A)—N(2A)—H(2AA) 106.7C(14A)—N(2A)—H(2AA) 106.7 C(13A)—N(2A)—H(2AA) 106.7 C(3A)—N(3A)—C(16A)114.4(3) C(3A)—N(3A)—C(15A) 111.3(4) C(16A)—N(3A)—C(15A) 110.1(4)C(3A)—N(3A)—H(3A) 106.8 C(16A)—N(3A)—H(3A) 106.8 C(15A)—N(3A)—H(3A)106.8 C(2A)—C(1A)—C(6A) 120.3(4) C(2A)—C(1A)—S(1A) 116.7(3)C(6A)—C(1A)—S(1A) 123.0(3) C(3A)—C(2A)—C(1A) 120.4(4) C(3A)—C(2A)—H(2A)119.8 C(1A)—C(2A)—H(2A) 119.8 C(2A)—C(3A)—C(4A) 120.6(4)C(2A)—C(3A)—N(3A) 120.2(4) C(4A)—C(3A)—N(3A) 119.1(4) C(3A)—C(4A)—C(5A)118.8(4) C(3A)—C(4A)—H(4A) 120.6 C(5A)—C(4A)—H(4A) 120.6C(6A)—C(5A)—C(4A) 121.8(4) C(6A)—C(5A)—H(5A) 119.1 C(4A)—C(5A)—H(5A)119.1 C(5A)—C(6A)—N(1A) 119.7(4) C(5A)—C(6A)—C(1A) 118.0(4)N(1A)—C(6A)—C(1A) 122.3(4) C(8A)—C(7A)—N(1A) 119.7(4) C(8A)—C(7A)—C(12A)118.4(4) N(1A)—C(7A)—C(12A) 121.9(4) C(7A)—C(8A)—C(9A) 121.9(4)C(7A)—C(8A)—H(8A) 119.0 C(9A)—C(8A)—H(8A) 119.0 C(10A)—C(9A)—C(8A)118.4(4) C(10A)—C(9A)—H(9A) 120.8 C(8A)—C(9A)—H(9A) 120.8C(11A)—C(10A)—C(9A) 120.9(4) C(11A)—C(10A)—N(2A) 117.9(4)C(9A)—C(10A)—N(2A) 121.2(4) C(10A)—C(11A)—C(12A) 120.4(4)C(10A)—C(11A)—H(11A) 119.8 C(12A)—C(11A)—H(11A) 119.8C(11A)—C(12A)—C(7A) 119.9(4) C(11A)—C(12A)—S(1A) 116.5(3)C(7A)—C(12A)—S(1A) 123.5(3) N(2A)—C(13A)—H(13A) 109.5N(2A)—C(13A)—H(13B) 109.5 H(13A)—C(13A)—H(13B) 109.5 N(2A)—C(13A)—H(13C)109.5 H(13A)—C(13A)—H(13C) 109.5 H(13B)—C(13A)—H(13C) 109.5N(2A)—C(14A)—H(14A) 109.5 N(2A)—C(14A)—H(14B) 109.5 H(14A)—C(14A)—H(14B)109.5 N(2A)—C(14A)—H(14C) 109.5 H(14A)—C(14A)—H(14C) 109.5H(14B)—C(14A)—H(14C) 109.5 N(3A)—C(15A)—H(15A) 109.5 N(3A)—C(15A)—H(15B)109.5 H(15A)—C(15A)—H(15B) 109.5 N(3A)—C(15A)—H(15C) 109.5H(15A)—C(15A)—H(15C) 109.5 H(15B)—C(15A)—H(15C) 109.5N(3A)—C(16A)—H(16A) 109.5 N(3A)—C(16A)—H(16B) 109.5 H(16A)—C(16A)—H(16B)109.5 N(3A)—C(16A)—H(16C) 109.5 H(16A)—C(16A)—H(16C) 109.5H(16B)—C(16A)—H(16C) 109.5 C(12B)—S(1B)—C(1B) 101.6(2) C(6B)—N(1B)—C(7B)125.1(4) C(6B)—N(1B)—H(1B) 117.4 C(7B)—N(1B)—H(1B) 117.4C(10B)—N(2B)—C(14B) 111.3(3) C(10B)—N(2B)—C(13B) 114.6(4)C(14B)—N(2B)—C(13B) 110.7(4) C(10B)—N(2B)—H(2BB) 106.5C(14B)—N(2B)—H(2BB) 106.5 C(13B)—N(2B)—H(2BB) 106.5 C(3B)—N(3B)—C(16B)113.0(3) C(3B)—N(3B)—C(15B) 111.8(3) C(16B)—N(3B)—C(15B) 111.1(4)C(3B)—N(3B)—H(3B) 106.9 C(16B)—N(3B)—H(3B) 106.9 C(15B)—N(3B)—H(3B)106.9 C(2B)—C(1B)—C(6B) 120.4(4) C(2B)—C(1B)—S(1B) 117.8(3)C(6B)—C(1B)—S(1B) 121.5(3) C(3B)—C(2B)—C(1B) 119.7(4) C(3B)—C(2B)—H(2B)120.2 C(1B)—C(2B)—H(2B) 120.2 C(2B)—C(3B)—C(4B) 121.1(4)C(2B)—C(3B)—N(3B) 120.3(4) C(4B)—C(3B)—N(3B) 118.7(4) C(5B)—C(4B)—C(3B)119.2(4) C(5B)—C(4B)—H(4B) 120.4 C(3B)—C(4B)—H(4B) 120.4C(4B)—C(5B)—C(6B) 121.1(4) C(4B)—C(5B)—H(5B) 119.5 C(6B)—C(5B)—H(5B)119.5 N(1B)—C(6B)—C(5B) 120.1(4) N(1B)—C(6B)—C(1B) 121.4(4)C(5B)—C(6B)—C(1B) 118.5(4) N(1B)—C(7B)—C(8B) 119.9(4) N(1B)—C(7B)—C(12B)121.8(4) C(8B)—C(7B)—C(12B) 118.3(4) C(9B)—C(8B)—C(7B) 121.6(4)C(9B)—C(8B)—H(8B) 119.2 C(7B)—C(8B)—H(8B) 119.2 C(8B)—C(9B)—C(10B)119.0(4) C(8B)—C(9B)—H(9B) 120.5 C(10B)—C(9B)—H(9B) 120.5C(9B)—C(10B)—C(11B) 121.4(4) C(9B)—C(10B)—N(2B) 120.8(4)C(11B)—C(10B)—N(2B) 117.6(4) C(10B)—C(11B)—C(12B) 119.5(4)C(10B)—C(11B)—H(11B) 120.3 C(12B)—C(11B)—H(11B) 120.3C(11B)—C(12B)—C(7B) 120.3(4) C(11B)—C(12B)—S(1B) 118.2(3)C(7B)—C(12B)—S(1B) 121.2(3) N(2B)—C(13B)—H(13D) 109.5N(2B)—C(13B)—H(13E) 109.5 H(13D)—C(13B)—H(13E) 109.5 N(2B)—C(13B)—H(13F)109.5 H(13D)—C(13B)—H(13F) 109.5 H(13E)—C(13B)—H(13F) 109.5N(2B)—C(14B)—H(14D) 109.5 N(2B)—C(14B)—H(14E) 109.5 H(14D)—C(14B)—H(14E)109.5 N(2B)—C(14B)—H(14F) 109.5 H(14D)—C(14B)—H(14F) 109.5H(14E)—C(14B)—H(14F) 109.5 N(3B)—C(15B)—H(15D) 109.5 N(3B)—C(15B)—H(15E)109.5 H(15D)—C(15B)—H(15E) 109.5 N(3B)—C(15B)—H(15F) 109.5H(15D)—C(15B)—H(15F) 109.5 H(15E)—C(15B)—H(15F) 109.5N(3B)—C(16B)—H(16D) 109.5 N(3B)—C(16B)—H(16E) 109.5 H(16D)—C(16B)—H(16E)109.5 N(3B)—C(16B)—H(16F) 109.5 H(16D)—C(16B)—H(16F) 109.5H(16E)—C(16B)—H(16F) 109.5 C(12C)—S(1C)—C(1C) 101.7(2) C(7C)—N(1C)—C(6C)125.4(4) C(7C)—N(1C)—H(1C) 117.3 C(6C)—N(1C)—H(1C) 117.3C(10C)—N(2C)—C(14C) 114.9(3) C(10C)—N(2C)—C(13C) 110.2(3)C(14C)—N(2C)—C(13C) 111.2(3) C(10C)—N(2C)—H(2CC) 106.7C(14C)—N(2C)—H(2CC) 106.7 C(13C)—N(2C)—H(2CC) 106.7 C(3C)—N(3C)—C(15C)111.2(4) C(3C)—N(3C)—C(16C) 112.9(4) C(15C)—N(3C)—C(16C) 111.5(4)C(3C)—N(3C)—H(3C) 106.9 C(15C)—N(3C)—H(3C) 106.9 C(16C)—N(3C)—H(3C)106.9 C(2C)—C(1C)—C(6C) 120.3(4) C(2C)—C(1C)—S(1C) 117.7(3)C(6C)—C(1C)—S(1C) 121.8(3) C(1C)—C(2C)—C(3C) 119.7(4) C(1C)—C(2C)—H(2C)120.2 C(3C)—C(2C)—H(2C) 120.2 C(2C)—C(3C)—C(4C) 121.8(4)C(2C)—C(3C)—N(3C) 118.6(4) C(4C)—C(3C)—N(3C) 119.5(4) C(3C)—C(4C)—C(5C)118.7(4) C(3C)—C(4C)—H(4C) 120.7 C(5C)—C(4C)—H(4C) 120.7C(4C)—C(5C)—C(6C) 120.9(4) C(4C)—C(5C)—H(5C) 119.5 C(6C)—C(5C)—H(5C)119.5 N(1C)—C(6C)—C(5C) 119.9(4) N(1C)—C(6C)—C(1C) 121.3(4)C(5C)—C(6C)—C(1C) 118.7(4) N(1C)—C(7C)—C(8C) 119.9(4) N(1C)—C(7C)—C(12C)122.0(4) C(8C)—C(7C)—C(12C) 118.1(4) C(9C)—C(8C)—C(7C) 121.5(4)C(9C)—C(8C)—H(8C) 119.2 C(7C)—C(8C)—H(8C) 119.2 C(8C)—C(9C)—C(10C)119.3(4) C(8C)—C(9C)—H(9C) 120.3 C(10C)—C(9C)—H(9C) 120.3C(11C)—C(10C)—C(9C) 120.5(4) C(11C)—C(10C)—N(2C) 117.5(4)C(9C)—C(10C)—N(2C) 121.9(4) C(12C)—C(11C)—C(10C) 120.4(4)C(12C)—C(11C)—H(11C) 119.8 C(10C)—C(11C)—H(11C) 119.8C(11C)—C(12C)—C(7C) 120.1(4) C(11C)—C(12C)—S(1C) 118.4(3)C(7C)—C(12C)—S(1C) 121.3(3) N(2C)—C(13C)—H(13G) 109.5N(2C)—C(13C)—H(13H) 109.5 H(13G)—C(13C)—H(13H) 109.5 N(2C)—C(13C)—H(13I)109.5 H(13G)—C(13C)—H(13I) 109.5 H(13H)—C(13C)—H(13I) 109.5N(2C)—C(14C)—H(14G) 109.5 N(2C)—C(14C)—H(14H) 109.5 H(14G)—C(14C)—H(14H)109.5 N(2C)—C(14C)—H(14I) 109.5 H(14G)—C(14C)—H(14I) 109.5H(14H)—C(14C)—H(14I) 109.5 N(3C)—C(15C)—H(15G) 109.5 N(3C)—C(15C)—H(15H)109.5 H(15G)—C(15C)—H(15H) 109.5 N(3C)—C(15C)—H(15I) 109.5H(15G)—C(15C)—H(15I) 109.5 H(15H)—C(15C)—H(15I) 109.5N(3C)—C(16C)—H(16G) 109.5 N(3C)—C(16C)—H(16H) 109.5 H(16G)—C(16C)—H(16H)109.5 N(3C)—C(16C)—H(16I) 109.5 H(16G)—C(16C)—H(16I) 109.5H(16H)—C(16C)—H(16I) 109.5 C(12D)—S(1D)—C(1D) 102.4(2) C(7D)—N(1D)—C(6D)126.5(4) C(7D)—N(1D)—H(1D) 116.7 C(6D)—N(1D)—H(1D) 116.7C(10D)—N(2D)—C(13D) 114.5(4) C(10D)—N(2D)—C(14D) 111.3(3)C(13D)—N(2D)—C(14D) 110.7(4) C(10D)—N(2D)—H(2D) 106.6 C(13D)—N(2D)—H(2D)106.6 C(14D)—N(2D)—H(2D) 106.6 C(3D)—N(3D)—C(15D) 111.2(4)C(3D)—N(3D)—C(16D) 114.3(4) C(15D)—N(3D)—C(16D) 111.2(4)C(3D)—N(3D)—H(3D) 106.5 C(15D)—N(3D)—H(3D) 106.5 C(16D)—N(3D)—H(3D)106.5 C(2D)—C(1D)—C(6D) 120.2(4) C(2D)—C(1D)—S(1D) 117.4(3)C(6D)—C(1D)—S(1D) 122.4(3) C(3D)—C(2D)—C(1D) 119.7(4) C(3D)—C(2D)—H(2DD)120.1 C(1D)—C(2D)—H(2DD) 120.1 C(2D)—C(3D)—C(4D) 120.7(4)C(2D)—C(3D)—N(3D) 120.7(4) C(4D)—C(3D)—N(3D) 118.4(4) C(3D)—C(4D)—C(5D)119.2(5) C(3D)—C(4D)—H(4D) 120.4 C(5D)—C(4D)—H(4D) 120.4C(6D)—C(5D)—C(4D) 121.5(4) C(6D)—C(5D)—H(5D) 119.2 C(4D)—C(5D)—H(5D)119.2 C(5D)—C(6D)—N(1D) 118.7(4) C(5D)—C(6D)—C(1D) 118.6(4)N(1D)—C(6D)—C(1D) 122.7(4) N(1D)—C(7D)—C(8D) 119.7(4) N(1D)—C(7D)—C(12D)121.5(4) C(8D)—C(7D)—C(12D) 118.7(4) C(9D)—C(8D)—C(7D) 121.8(4)C(9D)—C(8D)—H(8D) 119.1 C(7D)—C(8D)—H(8D) 119.1 C(10D)—C(9D)—C(8D)118.3(4) C(10D)—C(9D)—H(9D) 120.9 C(8D)—C(9D)—H(9D) 120.9C(9D)—C(10D)—C(11D) 121.7(4) C(9D)—C(10D)—N(2D) 121.0(4)C(11D)—C(10D)—N(2D) 117.1(4) C(12D)—C(11D)—C(10D) 119.5(4)C(12D)—C(11D)—H(11D) 120.3 C(10D)—C(11D)—H(11D) 120.3C(11D)—C(12D)—C(7D) 120.0(4) C(11D)—C(12D)—S(1D) 116.4(3)C(7D)—C(12D)—S(1D) 123.4(3) N(2D)—C(13D)—H(13J) 109.5N(2D)—C(13D)—H(13K) 109.5 H(13J)—C(13D)—H(13K) 109.5 N(2D)—C(13D)—H(13L)109.5 H(13J)—C(13D)—H(13L) 109.5 H(13K)—C(13D)—H(13L) 109.5N(2D)—C(14D)—H(14J) 109.5 N(2D)—C(14D)—H(14K) 109.5 H(14J)—C(14D)—H(14K)109.5 N(2D)—C(14D)—H(14L) 109.5 H(14J)—C(14D)—H(14L) 109.5H(14K)—C(14D)—H(14L) 109.5 N(3D)—C(15D)—H(15J) 109.5 N(3D)—C(15D)—H(15K)109.5 H(15J)—C(15D)—H(15K) 109.5 N(3D)—C(15D)—H(15L) 109.5H(15J)—C(15D)—H(15L) 109.5 H(15K)—C(15D)—H(15L) 109.5N(3D)—C(16D)—H(16J) 109.5 N(3D)—C(16D)—H(16K) 109.5 H(16J)—C(16D)—H(16K)109.5 N(3D)—C(16D)—H(16L) 109.5 H(16J)—C(16D)—H(16L) 109.5H(16K)—C(16D)—H(16L) 109.5 O(2)—S(2)—O(3) 118.5(17) O(2)—S(2)—O(1′)87.9(12) O(3)—S(2)—O(1′) 108.0(8) O(2)—S(2)—O(3′) 117.2(14)O(3)—S(2)—O(3′) 31.5(6) O(1′)—S(2)—O(3′) 138.3(6) O(2)—S(2)—O(2′)23.8(18) O(3)—S(2)—O(2′) 115.1(10) O(1′)—S(2)—O(2′) 110.6(8)O(3′)—S(2)—O(2′) 101.4(8) O(2)—S(2)—O(1) 111.9(14) O(3)—S(2)—O(1)65.7(9) O(1′)—S(2)—O(1) 42.6(5) O(3′)—S(2)—O(1) 95.8(7) O(2′)—S(2)—O(1)134.1(9) O(2)—S(2)—C(1S) 117.0(16) O(3)—S(2)—C(1S) 118.1(6)O(1′)—S(2)—C(1S) 99.1(4) O(3′)—S(2)—C(1S) 97.8(4) O(2′)—S(2)—C(1S)104.8(8) O(1)—S(2)—C(1S) 114.6(5) O(5)—S(3)—O(6) 113.5(2) O(5)—S(3)—O(4)112.5(2) O(6)—S(3)—O(4) 111.9(2) O(5)—S(3)—C(15S) 107.5(2)O(6)—S(3)—C(15S) 106.2(2) O(4)—S(3)—C(15S) 104.6(2) O(7)—S(4)—O(9)113.8(2) O(7)—S(4)—O(8) 113.4(2) O(9)—S(4)—O(8) 111.3(2) O(7)—S(4)—C(3S)106.0(2) O(9)—S(4)—C(3S) 106.6(2) O(8)—S(4)—C(3S) 104.9(2)O(11)—S(5)—O(12) 112.3(3) O(11)—S(5)—O(10) 114.1(3) O(12)—S(5)—O(10)109.9(2) O(11)—S(5)—C(5S) 107.4(2) O(12)—S(5)—C(5S) 107.2(2)O(10)—S(5)—C(5S) 105.5(2) O(14)—S(6)—O(15) 112.2(2) O(14)—S(6)—O(13)113.4(3) O(15)—S(6)—O(13) 111.4(2) O(14)—S(6)—C(9S) 105.6(2)O(15)—S(6)—C(9S) 108.3(2) O(13)—S(6)—C(9S) 105.4(3) O(17)—S(7)—O(18)114.0(2) O(17)—S(7)—O(16) 114.2(2) O(18)—S(7)—O(16) 110.7(2)O(17)—S(7)—C(7S) 105.8(3) O(18)—S(7)—C(7S) 105.3(2) O(16)—S(7)—C(7S)106.0(2) O(21)—S(8)—O(19) 114.3(2) O(21)—S(8)—O(20) 111.7(2)O(19)—S(8)—O(20) 111.95(19) O(21)—S(8)—C(11S) 106.3(2) O(19)—S(8)—C(11S)108.1(2) O(20)—S(8)—C(11S) 103.7(2) O(24)—S(9)—O(22) 113.23(19)O(24)—S(9)—O(23) 112.94(19) O(22)—S(9)—O(23) 111.07(18)O(24)—S(9)—C(13S) 106.1(2) O(22)—S(9)—C(13S) 107.7(2) O(23)—S(9)—C(13S)105.3(2) S(2)—O(1)—O(3) 52.7(7) S(2)—O(3)—O(1) 61.6(8) C(2S)—C(1S)—S(2)115.9(5) C(2S)—C(1S)—H(1S1) 108.3 S(2)—C(1S)—H(1S1) 108.3C(2S)—C(1S)—H(1S2) 108.3 S(2)—C(1S)—H(1S2) 108.3 H(1S1)—C(1S)—H(1S2)107.4 C(1S)—C(2S)—H(2S1) 109.5 C(1S)—C(2S)—H(2S2) 109.5H(2S1)—C(2S)—H(2S2) 109.5 C(1S)—C(2S)—H(2S3) 109.5 H(2S1)—C(2S)—H(2S3)109.5 H(2S2)—C(2S)—H(2S3) 109.5 C(4S)—C(3S)—S(4) 111.3(3)C(4S)—C(3S)—H(3S1) 109.4 S(4)—C(3S)—H(3S1) 109.4 C(4S)—C(3S)—H(3S2)109.4 S(4)—C(3S)—H(3S2) 109.4 H(3S1)—C(3S)—H(3S2) 108.0C(3S)—C(4S)—H(4S1) 109.5 C(3S)—C(4S)—H(4S2) 109.5 H(4S1)—C(4S)—H(4S2)109.5 C(3S)—C(4S)—H(4S3) 109.5 H(4S1)—C(4S)—H(4S3) 109.5H(4S2)—C(4S)—H(4S3) 109.5 C(6S)—C(5S)—S(5) 112.7(3) C(6S)—C(5S)—H(5S1)109.0 S(5)—C(5S)—H(5S1) 109.0 C(6S)—C(5S)—H(5S2) 109.0 S(5)—C(5S)—H(5S2)109.0 H(5S1)—C(5S)—H(5S2) 107.8 C(5S)—C(6S)—H(6S1) 109.5C(5S)—C(6S)—H(6S2) 109.5 H(6S1)—C(6S)—H(6S2) 109.5 C(5S)—C(6S)—H(6S3)109.5 H(6S1)—C(6S)—H(6S3) 109.5 H(6S2)—C(6S)—H(6S3) 109.5C(8S)—C(7S)—S(7) 110.6(4) C(8S)—C(7S)—H(7S1) 109.5 S(7)—C(7S)—H(7S1)109.5 C(8S)—C(7S)—H(7S2) 109.5 S(7)—C(7S)—H(7S2) 109.5H(7S1)—C(7S)—H(7S2) 108.1 C(7S)—C(8S)—H(8S1) 109.5 C(7S)—C(8S)—H(8S2)109.5 H(8S1)—C(8S)—H(8S2) 109.5 C(7S)—C(8S)—H(8S3) 109.5H(8S1)—C(8S)—H(8S3) 109.5 H(8S2)—C(8S)—H(8S3) 109.5 C(10S)—C(9S)—S(6)104.5(5) C(10S)—C(9S)—H(9S1) 110.8 S(6)—C(9S)—H(9S1) 110.8C(10S)—C(9S)—H(9S2) 110.8 S(6)—C(9S)—H(9S2) 110.8 H(9S1)—C(9S)—H(9S2)108.9 C(9S)—C(10S)—H(10A) 109.5 C(9S)—C(10S)—H(10B) 109.5H(10A)—C(10S)—H(10B) 109.5 C(9S)—C(10S)—H(10C) 109.5H(10A)—C(10S)—H(10C) 109.5 H(10B)—C(10S)—H(10C) 109.5 C(12S)—C(11S)—S(8)113.0(3) C(12S)—C(11S)—H(11E) 109.0 S(8)—C(11S)—H(11E) 109.0C(12S)—C(11S)—H(11F) 109.0 S(8)—C(11S)—H(11F) 109.0 H(11E)—C(11S)—H(11F)107.8 C(11S)—C(12S)—H(12A) 109.5 C(11S)—C(12S)—H(12B) 109.5H(12A)—C(12S)—H(12B) 109.5 C(11S)—C(12S)—H(12C) 109.5H(12A)—C(12S)—H(12C) 109.5 H(12B)—C(12S)—H(12C) 109.5 C(14S)—C(13S)—S(9)111.6(3) C(14S)—C(13S)—H(13M) 109.3 S(9)—C(13S)—H(13M) 109.3C(14S)—C(13S)—H(13N) 109.3 S(9)—C(13S)—H(13N) 109.3 H(13M)—C(13S)—H(13N)108.0 C(13S)—C(14S)—H(14M) 109.5 C(13S)—C(14S)—H(14N) 109.5H(14M)—C(14S)—H(14N) 109.5 C(13S)—C(14S)—H(14O) 109.5H(14M)—C(14S)—H(14O) 109.5 H(14N)—C(14S)—H(14O) 109.5 C(16S)—C(15S)—S(3)112.7(4) C(16S)—C(15S)—H(15M) 109.0 S(3)—C(15S)—H(15M) 109.0C(16S)—C(15S)—H(15N) 109.0 S(3)—C(15S)—H(15N) 109.0 H(15M)—C(15S)—H(15N)107.8 C(15S)—C(16S)—H(16M) 109.5 C(15S)—C(16S)—H(16N) 109.5H(16M)—C(16S)—H(16N) 109.5 C(15S)—C(16S)—H(16O) 109.5H(16M)—C(16S)—H(16O) 109.5 H(16N)—C(16S)—H(16O) 109.5 Symmetrytransformations used to generate equivalent atoms:

TABLE 4 Anisotropic displacement parameters (Å² × 10³) for LMT·2EsOH.The anisotropic displacement factor exponent takes the form: −2□²[h²a*²U¹¹ + . . . + 2 h k a* b* U¹²] U¹¹ U²² U³³ U²³ U¹³ U¹²  S(1A) 18(1)21(1) 121(2)  12(1) −15(1)  −2(1)  N(1A) 15(2) 24(2) 36(2)  1(2) 0(2)−3(2)  N(2A) 19(2) 20(2) 27(2)  5(2) 1(2) 0(2) N(3A) 17(2) 23(2) 27(2)−1(2) 3(2) 1(2) C(1A) 20(3) 22(2) 39(3)  3(2) −4(2)  −1(2)  C(2A) 17(2)24(2) 39(3)  2(2) −4(2)  −1(2)  C(3A) 22(2) 22(2) 25(2) −1(2) 7(2)−2(2)  C(4A) 17(2) 29(2) 31(3) −5(2) 7(2) 2(2) C(5A) 15(2) 28(2) 37(3)−6(2) 9(2) −6(2)  C(6A) 18(2) 23(2) 26(2) −2(2) 3(2) −3(2)  C(7A) 22(2)23(2) 25(2)  0(2) 5(2) −2(2)  C(8A) 21(2) 25(2) 21(2) −3(2) 6(2) −7(2) C(9A) 25(2) 22(2) 24(2)  2(2) 7(2) −3(2)   C(10A) 19(2) 24(2) 22(2) 3(2) 4(2) 2(2)  C(11A) 19(2) 23(2) 33(3)  2(2) 1(2) −8(2)   C(12A)20(3) 22(2) 42(3)  6(2) 2(2) −1(2)   C(13A) 28(3) 39(3) 30(3) 15(2) 0(2)−2(2)   C(14A) 34(3) 44(3) 37(3)  1(2) 7(2) 13(2)   C(15A) 96(5) 26(3)36(3)  3(2) 37(3)  2(3)  C(16A) 32(3) 26(2) 30(3) −9(2) 4(2) 6(2)  S(1B)17(1) 18(1) 39(1)  3(1) 7(1) −1(1)  N(1B) 21(2) 16(2) 25(2) −2(2) 8(2)−3(2)  N(2B) 29(2) 24(2) 20(2)  4(2) 1(2) 6(2) N(3B) 21(2) 19(2) 21(2)−2(2) 3(2) 2(2) C(1B) 17(2) 20(2) 18(2)  0(2) 5(2) 0(2) C(2B) 21(2)23(2) 16(2) −3(2) 3(2) −4(2)  C(3B) 22(2) 18(2) 18(2) −2(2) 6(2) 1(2)C(4B) 16(2) 23(2) 20(2) −1(2) 3(2) 0(2) C(5B) 16(2) 21(2) 21(2) −1(2)5(2) −4(2)  C(6B) 25(2) 20(2) 17(2)  0(2) 9(2) 1(2) C(7B) 22(2) 24(2)20(2) −3(2) 9(2) 0(2) C(8B) 29(3) 19(2) 27(2)  0(2) 17(2)  −2(2)  C(9B)32(3) 20(2) 25(2)  3(2) 16(2)  4(2)  C(10B) 31(3) 24(2) 14(2)  1(2) 7(2)9(2)  C(11B) 24(2) 23(2) 19(2) −2(2) 6(2) −1(2)   C(12B) 28(3) 18(2)20(2)  0(2) 9(2) 2(2)  C(13B) 53(3) 30(3) 26(3) 14(2) 11(2)  17(2)  C(14B) 22(2) 30(2) 27(3)  1(2) 4(2) 6(2)  C(15B) 42(3) 22(2) 35(3) 3(2) 15(2)  3(2)  C(16B) 39(3) 30(3) 25(3) −8(2) 2(2) 10(2)   S(1C)16(1) 21(1) 34(1)  2(1) 6(1) 0(1) N(1C) 15(2) 22(2) 31(2) −8(2) 4(2)−5(2)  N(2C) 16(2) 19(2) 23(2)  0(2) 6(2) 0(1) N(3C) 21(2) 28(2) 27(2) 1(2) 7(2) 5(2) C(1C) 16(2) 25(2) 15(2) −1(2) 2(2) 0(2) C(2C) 20(2)24(2) 17(2) −2(2) 6(2) −1(2)  C(3C) 21(2) 20(2) 19(2)  1(2) 6(2) 5(2)C(4C) 20(2) 34(3) 20(2) −2(2) 7(2) 4(2) C(5C) 20(2) 26(2) 20(2) −1(2)6(2) −1(2)  C(6C) 22(2) 24(2) 15(2) −4(2) 7(2) −1(2)  C(7C) 18(2) 29(2)17(2) −4(2) 6(2) 2(2) C(8C) 17(2) 22(2) 27(2) −8(2) 9(2) −6(2)  C(9C)21(2) 20(2) 22(2) −3(2) 10(2)  −2(2)   C(10C) 14(2) 25(2) 18(2) −3(2)5(2) 1(2)  C(11C) 15(2) 24(2) 21(2) −5(2) 6(2) −7(2)   C(12C) 18(2)19(2) 21(2) −4(2) 7(2) 2(2)  C(13C) 17(2) 30(2) 29(3)  2(2) 9(2) 3(2) C(14C) 23(2) 23(2) 23(2)  2(2) 7(2) −2(2)   C(15C) 46(3) 45(3) 56(4)22(3) 29(3)  11(3)   C(16C) 38(3) 33(3) 46(3) −18(2)  −4(3)  8(2)  S(1D)25(1) 23(1) 46(1) −3(1) 13(1)  −4(1)  N(1D) 26(2) 23(2) 36(2) −4(2)11(2)  −4(2)  N(2D) 22(2) 35(2) 23(2)  2(2) 7(2) 2(2) N(3D) 27(2) 24(2)34(2)  0(2) 16(2)  2(2) C(1D) 28(3) 28(2) 20(2) −3(2) 10(2)  0(2) C(2D)30(3) 29(2) 23(2) −5(2) 13(2)  −4(2)  C(3D) 33(3) 24(2) 24(2) −1(2)13(2)  1(2) C(4D) 27(3) 31(3) 33(3)  0(2) 10(2)  −1(2)  C(5D) 26(3)29(3) 36(3) −6(2) 8(2) −6(2)  C(6D) 29(3) 24(2) 19(2) −3(2) 10(2)  0(2)C(7D) 23(2) 27(2) 16(2)  0(2) 9(2) −1(2)  C(8D) 30(3) 27(2) 19(2)  0(2)15(2)  −3(2)  C(9D) 29(3) 29(2) 16(2)  3(2) 9(2) −2(2)   C(10D) 24(2)30(2) 17(2) −2(2) 8(2) 0(2)  C(11D) 26(3) 32(2) 17(2) −6(2) 7(2) −6(2)  C(12D) 28(3) 24(2) 18(2) −5(2) 11(2)  −1(2)   C(13D) 33(3) 60(4) 33(3)19(3) 11(2)  10(3)   C(14D) 30(3) 33(3) 32(3)  3(2) 16(2)  5(2)  C(15D)51(3) 34(3) 41(3)  6(2) 23(3)  10(2)   C(16D) 35(3) 26(2) 47(3) −12(2) 19(3)  −3(2)  S(2)  36(1) 27(1) 45(1)  9(1) 18(1)  2(1) S(3)  23(1)22(1) 24(1) −3(1) 4(1) −1(1)  S(4)  16(1) 23(1) 40(1) −4(1) 4(1) −1(1) S(5)  23(1) 24(1) 41(1)  8(1) 12(1)  4(1) S(6)  42(1) 19(1) 29(1)  0(1)9(1) −2(1)  S(7)  31(1) 28(1) 39(1)  4(1) 12(1)  1(1) S(8)  20(1) 19(1)24(1)  0(1) 4(1) −1(1)  S(9)  23(1) 23(1) 27(1) −1(1) 7(1) 4(1) O(1) 67(4) 53(4) 52(5) 19(4) 29(4)  10(4)  O(2)  56(6) 25(9) 107(14) 17(8)13(8)  −20(7)  O(3)  55(4) 54(4) 57(5) 23(4) 25(4)  8(3) O(1′)  79(5)68(4) 48(4) 25(3) 39(4)  31(4)  O(2′)  53(4) 20(7) 109(13) 14(6) 9(7)−4(5)  O(3′)  35(3) 71(5) 68(5) 41(4) 21(4)  9(3) O(4)  26(2) 26(2)44(2)  3(2) 7(2) 2(1) O(5)  40(2) 33(2) 35(2) −10(2)  0(2) −8(2)  O(6) 23(2) 31(2) 26(2) −2(1) 9(1) −6(1)  O(7)  27(2) 27(2) 72(3) 11(2) 11(2) 0(2) O(8)  16(2) 42(2) 57(2) −18(2)  −3(2)  −2(2)  O(9)  24(2) 29(2)38(2) −2(1) 14(2)  −3(1)  O(10)  43(2) 49(2) 33(2) 12(2) 18(2)  30(2) O(11)  37(2) 48(2) 95(4) 34(2) 4(2) −11(2)  O(12)  78(3) 30(2) 34(2)−2(2) 28(2)  3(2) O(13)  52(3) 72(3) 56(3) −16(2)  35(2)  −33(2)  O(14) 94(3) 25(2) 35(2) −2(2) 3(2) 13(2)  O(15)  37(2) 40(2) 35(2) −9(2) 1(2)5(2) O(16)  23(2) 37(2) 36(2) −16(2)  13(2)  −8(1)  O(17)  59(3) 36(2)68(3) 23(2) 21(2)  −5(2)  O(18)  28(2) 52(2) 32(2) −9(2) 9(2) 0(2)O(19)  33(2) 19(2) 34(2) −1(1) 7(2) −2(1)  O(20)  20(2) 24(2) 45(2)−4(2) −1(2)  −2(1)  O(21)  32(2) 34(2) 28(2)  3(1) 14(2)  2(1) O(22) 26(2) 27(2) 24(2) −2(1) 9(1) −2(1)  O(23)  26(2) 23(2) 39(2)  3(1) 9(2)8(1) O(24)  26(2) 25(2) 42(2) −5(2) 8(2) −4(1)  C(1S)  60(4) 50(4) 56(4)14(3) 5(3) 6(3) C(2S)  48(3) 52(3) 36(3) 10(3) 19(3)  −2(3)  C(3S) 20(2) 37(3) 30(3) −4(2) 6(2) 5(2) C(4S)  32(3) 57(3) 28(3) 14(2) 12(2) 8(2) C(5S)  23(3) 31(3) 50(3) 11(2) 9(2) 8(2) C(6S)  37(3) 32(3) 26(3) 6(2) 13(2)  3(2) C(7S)  32(3) 47(3) 38(3) −2(2) 14(2)  1(2) C(8S) 48(4) 22(3) 73(4)  9(3) 8(3) −3(2)  C(9S)  45(3) 31(3) 43(3) 11(2)24(3)  7(2)  C(10S) 118(8)  88(6) 104(7)  −11(5)  58(6)  −22(6)   C(11S)30(3) 25(2) 30(3)  3(2) 14(2)  −2(2)   C(12S) 40(3) 40(3) 30(3)  3(2)11(2)  2(2)  C(13S) 29(3) 25(2) 26(2)  0(2) 11(2)  1(2)  C(14S) 35(3)32(3) 37(3) −2(2) 18(2)  −2(2)   C(15S) 38(3) 39(3) 35(3)  2(2) 19(2) −2(2)   C(16S) 51(4) 50(3) 63(4)  5(3) 40(3)  4(3)

TABLE 5 Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for LMT·2EsOH. x y z U(eq) H(1AA) −585 2958 101935 H(2AA) 1142 2510 2886 30 H(3A) −993 5379 587 30 H(2A) −112 4894 148238 H(4A) −1173 4519 414 32 H(5A) −1024 3623 616 33 H(8A) −239 2190 141229 H(9A) 327 1779 2005 30 H(11A) 801 3220 2506 34 H(13A) 956 2107 366855 H(13B) 1268 1770 3586 55 H(13C) 853 1592 3162 55 H(14A) 950 1606 204162 H(14B) 1350 1846 2447 62 H(14C) 1048 2153 1770 62 H(15A) −399 5529288 75 H(15B) −759 5883 −21 75 H(15C) −775 5281 −280 75 H(16A) −638 56071762 49 H(16B) −652 6075 1222 49 H(16C) −306 5694 1555 49 H(1B) 2048 25885 25 H(2BB) 3779 −238 2945 33 H(3B) 1492 2364 765 27 H(2B) 2408 20011444 26 H(4B) 1368 1494 658 26 H(5B) 1566 616 765 25 H(8B) 2428 −7041523 28 H(9B) 3012 −1036 2140 29 H(11B) 3416 453 2358 28 H(13D) 3579−889 3507 57 H(13E) 3953 −1033 3472 57 H(13F) 3582 −1293 2905 57 H(14D)3650 −952 1829 43 H(14E) 4042 −744 2383 43 H(14F) 3758 −345 1811 43H(15D) 2099 2713 612 50 H(15E) 1729 3039 363 50 H(15F) 1737 2500 −42 50H(16D) 1800 2460 1997 53 H(16E) 1763 3014 1591 53 H(16F) 2137 2692 188353 H(1C) 4627 2587 1195 30 H(2CC) 6358 2390 3285 24 H(3C) 4060 4939 95032 H(2C) 4971 4574 1649 25 H(4C) 3932 4060 845 30 H(5C) 4137 3182 100627 H(8C) 5015 1871 1821 26 H(9C) 5601 1557 2480 25 H(11C) 5987 3052 267325 H(13G) 6256 1619 2248 39 H(13H) 6636 1887 2761 39 H(13I) 6327 22302142 39 H(14G) 6168 1769 3891 36 H(14H) 6544 1621 3871 36 H(14I) 61771339 3323 36 H(15G) 4636 5189 631 70 H(15H) 4280 5544 406 70 H(15I) 42524970 55 70 H(16G) 4450 5092 2174 70 H(16H) 4333 5614 1683 70 H(16I) 47235354 1894 70 H(1D) 7088 320 1462 34 H(2D) 8828 −56 3133 33 H(3D) 66182711 1123 32 H(2DD) 7510 2283 1724 32 H(4D) 6459 1833 909 38 H(5D) 6639950 1112 38 H(8D) 7452 −429 1911 28 H(9D) 8028 −812 2416 30 H(11D) 8478655 2709 31 H(13J) 8654 −656 3792 65 H(13K) 9004 −828 3683 65 H(13L)8618 −1085 3187 65 H(14J) 8622 −820 2060 46 H(14K) 9023 −587 2493 46H(14L) 8707 −220 1935 46 H(15J) 7042 2819 377 61 H(15K) 6720 3214 295 61H(15L) 6632 2612 33 61 H(16J) 7101 2976 2179 54 H(16K) 6965 3437 1582 54H(16L) 7320 3101 1708 54 H(1S1) 354 568 117 74 H(1S2) 34 756 315 74H(2S1) 348 1552 757 67 H(2S2) 230 1466 −93 67 H(2S3) 641 1363 479 67H(3S1) 2296 4750 523 36 H(3S2) 2109 4415 −213 36 H(4S1) 1692 5133 −74959 H(4S2) 2102 5315 −517 59 H(4S3) 1901 5469 −21 59 H(5S1) 2641 3235 24144 H(5S2) 2548 3575 803 44 H(6S1) 3029 4152 984 48 H(6S2) 2760 4126 13748 H(6S3) 3134 3806 444 48 H(7S1) 5283 2814 −129 47 H(7S2) 5465 3051 67947 H(8S1) 5796 3675 290 81 H(8S2) 5368 3758 −148 81 H(8S3) 5587 3453−521 81 H(9S1) 5428 806 551 46 H(9S2) 5579 1289 1119 46 H(10A) 6182 9811462 150 H(10B) 5993 1119 613 150 H(10C) 6028 516 875 150 H(11E) 81093705 840 33 H(11F) 8195 3253 387 33 H(12A) 7581 2956 −55 57 H(12B) 76053553 −295 57 H(12C) 7500 3438 363 57 H(13M) 8341 797 1242 33 H(13N) 81181177 569 33 H(14M) 7744 447 −41 52 H(14N) 7716 589 698 52 H(14O) 7955 95671 52 H(15M) 272 2476 −63 44 H(15N) 475 2669 763 44 H(16M) 317 3544 40774 H(16N) −43 3188 97 74 H(16O) 135 3371 −430 74

Crystallographic Data for LMT.2MsOH (FIG. 17 c)

TABLE 1 Crystal data and structure refinement for LMT·2MsOH.Identification code 64412SC171 Empirical formula C18H27N3O6S3 Formulaweight 477.61 Temperature 150(2) K Wavelength 0.71073 Å Crystal systemTriclinic Space group P-1 Unit cell dimensions a = 11.6401(6) Å α =104.682(2)°. b = 12.0744(6) Å β = 92.386(2)°. c = 18.4846(9) Å γ =116.151(2)°. Volume 2220.42(19) Å³ Z 4 Density (calculated) 1.429 Mg/m³Absorption coefficient 0.374 mm⁻¹ F(000) 1008 Crystal size 0.30 × 0.18 ×0.04 mm³ Theta range for data collection 1.16 to 27.57°. Index ranges−15 <= h <= 15, −15 <= k <= 15, −24 <= l <= 24 Reflections collected42564 Independent reflections 10184 [R(int) = 0.0662] Completeness totheta = 25.00° 99.6% Absorption correction Semi-empirical fromequivalents Max. and min. transmission 0.9852 and 0.8962 Refinementmethod Full-matrix least-squares on F² Data/restraints/parameters10184/198/552 Goodness-of-fit on F² 1.071 Final R indices [I >2sigma(I)] R1 = 0.0593, wR2 = 0.1399 R indices (all data) R1 = 0.0909,wR2 = 0.1566 Largest diff. peak and hole 1.192 and −0.905 e · Å⁻³

TABLE 2 Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for eul1_0m. U(eq) is defined as one third of thetrace of the orthogonalized U^(ij) tensor. x y z U(eq) C(1A) 2847(3)7453(4) 3069(2) 28(1) C(2A) 2545(3) 8117(3) 2643(2) 27(1) C(3A) 3528(3)9173(4) 2496(2) 29(1) C(4A) 4823(3) 9566(4) 2760(2) 37(1) C(5A) 5121(3)8863(4) 3154(2) 39(1) C(6A) 4156(3) 7809(4) 3317(2) 34(1) C(7A) 3630(5)5911(5) 3768(2) 48(1) C(8A) 4139(5) 5179(5) 4015(2) 55(1) C(9A) 3314(5)4011(5) 4119(2) 58(1) C(10A) 1978(5) 3531(5) 3953(2) 52(1) C(11A)1451(5) 4217(4) 3678(2) 45(1) C(12A) 2292(4) 5408(4) 3601(2) 42(1)C(13A) 3947(4) 10426(4) 1555(2) 39(1) C(14A) 3035(4) 10981(4) 2698(2)36(1) C(15A) 479(5) 2617(4) 4788(2) 56(1) C(16A) 422(9) 1431(7) 3338(3)21(2) C(16′) −175(7) 1295(6) 3509(4) 38(2) C(1B) 1734(3) 3733(3) 1573(2)20(1) C(2B) 467(3) 2802(3) 1532(2) 20(1) C(3B) −556(3) 2949(3) 1228(2)20(1) C(4B) −328(3) 4011(3) 986(2) 21(1) C(5B) 938(3) 4959(3) 1054(2)22(1) C(6B) 1992(3) 4819(3) 1335(2) 21(1) C(7B) 4382(3) 5861(3) 1707(2)21(1) C(8B) 5559(3) 6955(3) 1766(2) 22(1) C(9B) 6724(3) 7111(3) 2126(2)22(1) C(10B) 6691(3) 6167(3) 2435(2) 20(1) C(11B) 5535(3) 5073(3)2382(2) 20(1) C(12B) 4385(3) 4907(3) 2011(2) 20(1) C(13B) −2276(3)1245(3) 1673(2) 27(1) C(14B) −2081(3) 892(3) 317(2) 27(1) C(15B) 8932(3)7573(3) 3209(2) 26(1) C(16B) 8431(3) 5579(3) 2180(2) 29(1) C(1S) 3536(4)59(4) 4695(2) 38(1) C(2S) 8797(4) 7948(3) 434(2) 33(1) C(3S) 5403(4)3853(4) 327(2) 40(1) C(4S) 6718(6) 3037(5) 4356(2) 67(2) N(1A) 4480(3)7134(4) 3726(2) 46(1) N(2A) 3126(3) 9915(3) 2118(2) 29(1) N(3A) 1056(5)2317(4) 4117(2) 68(1) N(1B) 3245(2) 5734(3) 1338(2) 24(1) N(2B) −1895(2)1871(3) 1060(1) 21(1) N(3B) 7903(2) 6237(2) 2774(2) 21(1) O(1S) 1876(5)153(5) 5602(3) 31(1) O(2S) 1457(5) 367(5) 4352(3) 27(1) O(3S) 3166(4)2156(4) 5329(3) 28(1) O(2S′) 3198(4) 1901(4) 4683(3) 26(1) O(1S′)2515(5) 924(5) 5683(3) 25(1) O(3S′) 1291(5) −191(6) 4380(3) 27(1) O(4S)10898(2) 10147(2) 1042(1) 34(1) O(5S) 9224(3) 9462(3) 1796(2) 40(1)O(6S) 10527(2) 8353(2) 1521(2) 39(1) O(7S) 6954(2) 2997(3) −257(1) 34(1)O(8S) 6130(2) 2435(2) 845(1) 31(1) O(9S) 4703(3) 1484(2) −383(2) 44(1)O(10S) 7552(3) 4905(3) 3786(2) 53(1) O(11S) 8403(4) 3384(4) 3483(2)73(1) O(12S) 6252(3) 2791(3) 2924(2) 57(1) S(1) 2478(1) 713(1) 4948(1)30(1) S(2) 9944(1) 9074(1) 1257(1) 23(1) S(3) 5818(1) 2588(1) 111(1)22(1) S(4) 7286(1) 3562(1) 3574(1) 26(1) S(1A) 1567(1) 6322(1) 3376(1)32(1) S(1B) 2994(1) 3393(1) 1838(1) 26(1)

TABLE 3 Bond lengths [Å] and angles [°] for eul1_0m. C(1A)—C(2A)1.390(5) C(1A)—C(6A) 1.408(4) C(1A)—S(1A) 1.753(4) C(2A)—C(3A) 1.388(5)C(2A)—H(2A) 0.9500 C(3A)—C(4A) 1.388(5) C(3A)—N(2A) 1.472(5) C(4A)—C(5A)1.387(6) C(4A)—H(4A) 0.9500 C(5A)—C(6A) 1.390(6) C(5A)—H(5A) 0.9500C(6A)—N(1A) 1.393(5) C(7A)—C(12A) 1.386(6) C(7A)—N(1A) 1.394(6)C(7A)—C(8A) 1.409(5) C(8A)—C(9A) 1.380(7) C(8A)—H(8A) 0.9500C(9A)—C(10A) 1.387(7) C(9A)—H(9A) 0.9500 C(10A)—C(11A) 1.399(5)C(10A)—N(3A) 1.502(7) C(11A)—C(12A) 1.386(7) C(11A)—H(11A) 0.9500C(12A)—S(1A) 1.768(3) C(13A)—N(2A) 1.506(4) C(13A)—H(13A) 0.9800C(13A)—H(13B) 0.9800 C(13A)—H(13C) 0.9800 C(14A)—N(2A) 1.499(5)C(14A)—H(14A) 0.9800 C(14A)—H(14B) 0.9800 C(14A)—H(14C) 0.9800C(15A)—N(3A) 1.477(6) C(15A)—H(15A) 0.9800 C(15A)—H(15B) 0.9800C(15A)—H(15C) 0.9800 C(16A)—N(3A) 1.482(6) C(16A)—H(16H) 0.9800C(16A)—H(16I) 0.9800 C(16A)—H(16J) 0.9800 C(16′)—N(3A) 1.563(6)C(16′)—H(16K) 0.9800 C(16′)—H(16L) 0.9800 C(16′)—H(16M) 0.9800C(1B)—C(2B) 1.390(4) C(1B)—C(6B) 1.398(4) C(1B)—S(1B) 1.770(3)C(2B)—C(3B) 1.394(4) C(2B)—H(2B) 0.9500 C(3B)—C(4B) 1.384(4) C(3B)—N(2B)1.479(4) C(4B)—C(5B) 1.386(4) C(4B)—H(4B) 0.9500 C(5B)—C(6B) 1.407(4)C(5B)—H(5B) 0.9500 C(6B)—N(1B) 1.387(4) C(7B)—N(1B) 1.391(4) C(7B)—C(8B)1.397(4) C(7B)—C(12B) 1.405(4) C(8B)—C(9B) 1.398(4) C(8B)—H(8B) 0.9500C(9B)—C(10B) 1.385(4) C(9B)—H(9B) 0.9500 C(10B)—C(11B) 1.387(4)C(10B)—N(3B) 1.478(4) C(11B)—C(12B) 1.386(4) C(11B)—H(11B) 0.9500C(12B)—S(1B) 1.766(3) C(13B)—N(2B) 1.494(4) C(13B)—H(13D) 0.9800C(13B)—H(13E) 0.9800 C(13B)—H(13F) 0.9800 C(14B)—N(2B) 1.503(4)C(14B)—H(14D) 0.9800 C(14B)—H(14E) 0.9800 C(14B)—H(14F) 0.9800C(15B)—N(3B) 1.497(4) C(15B)—H(15D) 0.9800 C(15B)—H(15E) 0.9800C(15B)—H(15F) 0.9800 C(16B)—N(3B) 1.503(4) C(16B)—H(16A) 0.9800C(16B)—H(16B) 0.9800 C(16B)—H(16C) 0.9800 C(1S)—S(1) 1.755(4)C(1S)—H(1S1) 0.9800 C(1S)—H(1S2) 0.9800 C(1S)—H(1S3) 0.9800 C(2S)—S(2)1.768(3) C(2S)—H(2S1) 0.9800 C(2S)—H(2S2) 0.9800 C(2S)—H(2S3) 0.9800C(3S)—S(3) 1.755(4) C(3S)—H(3S1) 0.9800 C(3S)—H(3S2) 0.9800 C(3S)—H(3S3)0.9800 C(4S)—S(4) 1.762(4) C(4S)—H(4S1) 0.9800 C(4S)—H(4S2) 0.9800C(4S)—H(4S3) 0.9800 N(1A)—H(1A) 0.8800 N(2A)—H(2A1) 0.9300 N(3A)—H(3A)0.9300 N(1B)—H(1B) 0.8800 N(2B)—H(2B1) 0.9300 N(3B)—H(3B) 0.9300O(1S)—S(1) 1.573(5) O(2S)—S(1) 1.422(5) O(3S)—S(1) 1.508(5) O(2S′)—S(1)1.528(5) O(1S′)—S(1) 1.312(5) O(3S′)—S(1) 1.473(5) O(4S)—S(2) 1.449(2)O(5S)—S(2) 1.447(3) O(6S)—S(2) 1.472(2) O(7S)—S(3) 1.458(2) O(8S)—S(3)1.467(2) O(9S)—S(3) 1.433(3) O(10S)—S(4) 1.451(3) O(11S)—S(4) 1.417(3)O(12S)—S(4) 1.443(3) C(2A)—C(1A)—C(6A) 119.8(3) C(2A)—C(1A)—S(1A)117.7(2) C(6A)—C(1A)—S(1A) 122.0(3) C(3A)—C(2A)—C(1A) 120.3(3)C(3A)—C(2A)—H(2A) 119.9 C(1A)—C(2A)—H(2A) 119.9 C(2A)—C(3A)—C(4A)120.5(4) C(2A)—C(3A)—N(2A) 116.9(3) C(4A)—C(3A)—N(2A) 122.3(3)C(5A)—C(4A)—C(3A) 118.9(4) C(5A)—C(4A)—H(4A) 120.5 C(3A)—C(4A)—H(4A)120.5 C(4A)—C(5A)—C(6A) 121.7(3) C(4A)—C(5A)—H(5A) 119.1C(6A)—C(5A)—H(5A) 119.1 C(5A)—C(6A)—N(1A) 120.6(3) C(5A)—C(6A)—C(1A)118.6(4) N(1A)—C(6A)—C(1A) 120.8(4) C(12A)—C(7A)—N(1A) 122.0(3)C(12A)—C(7A)—C(8A) 118.7(5) C(1A)—C(7A)—C(8A) 119.3(4) C(9A)—C(8A)—C(7A)120.2(5) C(9A)—C(8A)—H(8A) 119.9 C(7A)—C(8A)—H(8A) 119.9C(8A)—C(9A)—C(10A) 120.1(4) C(8A)—C(9A)—H(9A) 120.0 C(10A)—C(9A)—H(9A)120.0 C(9A)—C(10A)—C(11A) 120.6(5) C(9A)—C(10A)—N(3A) 121.4(4)C(11A)—C(10A)—N(3A) 117.8(4) C(12A)—C(11A)—C(10A) 118.5(4)C(12A)—C(11A)—H(11A) 120.7 C(10A)—C(11A)—H(11A) 120.7C(7A)—C(12A)—C(11A) 121.8(4) C(7A)—C(12A)—S(1A) 121.7(4)C(11A)—C(12A)—S(1A) 116.2(3) N(2A)—C(13A)—H(13A) 109.5N(2A)—C(13A)—H(13B) 109.5 H(13A)—C(13A)—H(13B) 109.5 N(2A)—C(13A)—H(13C)109.5 H(13A)—C(13A)—H(13C) 109.5 H(13B)—C(13A)—H(13C) 109.5N(2A)—C(14A)—H(14A) 109.5 N(2A)—C(14A)—H(14B) 109.5 H(14A)—C(14A)—H(14B)109.5 N(2A)—C(14A)—H(14C) 109.5 H(14A)—C(14A)—H(14C) 109.5H(14B)—C(14A)—H(14C) 109.5 N(3A)—C(15A)—H(15A) 109.5 N(3A)—C(15A)—H(15B)109.5 H(15A)—C(15A)—H(15B) 109.5 N(3A)—C(15A)—H(15C) 109.5H(15A)—C(15A)—H(15C) 109.5 H(15B)—C(15A)—H(15C) 109.5N(3A)—C(16A)—H(16H) 109.5 N(3A)—C(16A)—H(16I) 109.5 N(3A)—C(16A)—H(16J)109.5 N(3A)—C(16′)—H(16K) 109.5 N(3A)—C(16′)—H(16L) 109.5H(16K)—C(16′)—H(16L) 109.5 N(3A)—C(16′)—H(16M) 109.5 H(16K)—C(16)—H(16M)109.5 H(16L)—C(16′)—H(16M) 109.5 C(2B)—C(1B)—C(6B) 121.3(3)C(2B)—C(1B)—S(1B) 116.9(2) C(6B)—C(1B)—S(1B) 121.5(2) C(1B)—C(2B)—C(3B)118.7(3) C(1B)—C(2B)—H(2B) 120.7 C(3B)—C(2B)—H(2B) 120.7C(4B)—C(3B)—C(2B) 121.2(3) C(4B)—C(3B)—N(2B) 118.7(3) C(2B)—C(3B)—N(2B)119.5(3) C(3B)—C(4B)—C(5B) 119.7(3) C(3B)—C(4B)—H(4B) 120.1C(5B)—C(4B)—H(4B) 120.1 C(4B)—C(5B)—C(6B) 120.4(3) C(4B)—C(5B)—H(5B)119.8 C(6B)—C(5B)—H(5B) 119.8 N(1B)—C(6B)—C(1B) 122.5(3)N(1B)—C(6B)—C(5B) 118.7(3) C(1B)—C(6B)—C(5B) 118.7(3) N(1B)—C(7B)—C(8B)119.3(3) N(1B)—C(7B)—C(12B) 121.7(3) C(8B)—C(7B)—C(12B) 119.0(3)C(7B)—C(8B)—C(9B) 120.9(3) C(7B)—C(8B)—H(8B) 119.5 C(9B)—C(8B)—H(8B)119.5 C(10B)—C(9B)—C(8B) 118.7(3) C(10B)—C(913)—H(9B) 120.6C(8B)—C(9B)—H(9B) 120.6 C(9B)—C(10B)—C(11B) 121.4(3) C(9B)—C(10B)—N(3B)121.0(3) C(11B)—C(10B)—N(3B) 117.4(3) C(12B)—C(11B)—C(10B) 119.7(3)C(12B)—C(11B)—H(11B) 120.1 C(10B)—C(11B)—H(11B) 120.1C(11B)—C(12B)—C(7B) 120.2(3) C(11B)—C(12B)—S(1B) 117.6(2)C(7B)—C(12B)—S(1B) 121.8(2) N(2B)—C(13B)—H(13D) 109.5N(2B)—C(13B)—H(13E) 109.5 H(13D)—C(13B)—H(13E) 109.5 N(2B)—C(13B)—H(13F)109.5 H(13D)—C(13B)—H(13F) 109.5 H(13E)—C(13B)—H(13F) 109.5N(2B)—C(14B)—H(14D) 109.5 N(2B)—C(14B)—H(14E) 109.5 H(14D)—C(14B)—H(14E)109.5 N(2B)—C(14B)—H(14F) 109.5 H(14D)—C(14B)—H(14F) 109.5H(14E)—C(14B)—H(14F) 109.5 N(3B)—C(15B)—H(15D) 109.5 N(3B)—C(15B)—H(15E)109.5 H(15D)—C(15B)—H(15E) 109.5 N(3B)—C(15B)—H(15F) 109.5H(15D)—C(15B)—H(15F) 109.5 H(15E)—C(15B)—H(15F) 109.5N(3B)—C(16B)—H(16A) 109.5 N(3B)—C(16B)—H(16B) 109.5 H(16A)—C(16B)—H(16B)109.5 N(3B)—C(16B)—H(16C) 109.5 H(16A)—C(16B)—H(16C) 109.5H(16B)—C(16B)—H(16C) 109.5 S(1)—C(1S)—H(1S1) 109.5 S(1)—C(1S)—H(1S2)109.5 H(1S1)—C(1S)—H(1S2) 109.5 S(1)—C(1S)—H(1S3) 109.5H(1S1)—C(1S)—H(1S3) 109.5 H(1S2)—C(1S)—H(1S3) 109.5 S(2)—C(2S)—H(2S1)109.5 S(2)—C(2S)—H(2S2) 109.5 H(2S1)—C(2S)—H(2S2) 109.5S(2)—C(2S)—H(2S3) 109.5 H(2S1)—C(2S)—H(2S3) 109.5 H(2S2)—C(2S)—H(2S3)109.5 S(3)—C(3S)—H(3S1) 109.5 S(3)—C(3S)—H(3S2) 109.5H(3S1)—C(3S)—H(3S2) 109.5 S(3)—C(3S)—H(3S3) 109.5 H(3S1)—C(3S)—H(3S3)109.5 H(3S2)—C(3S)—H(3S3) 109.5 S(4)—C(4S)—H(4S1) 109.5S(4)—C(4S)—H(4S2) 109.5 H(4S1)—C(4S)—H(4S2) 109.5 S(4)—C(4S)—H(4S3)109.5 H(4S1)—C(4S)—H(4S3) 109.5 H(4S2)—C(4S)—H(4S3) 109.5C(6A)—N(1A)—C(7A) 124.8(3) C(6A)—N(1A)—H(1A) 117.6 C(7A)—N(1A)—H(1A)117.6 C(3A)—N(2A)—C(14A) 110.2(3) C(3A)—N(2A)—C(13A) 114.9(3)C(14A)—N(2A)—C(13A) 111.1(3) C(3A)—N(2A)—H(2A1) 106.7C(14A)—N(2A)—H(2A1) 106.7 C(13A)—N(2A)—H(2A1) 106.7 C(15A)—N(3A)—C(10A)111.1(3) C(15A)—N(3A)—C(16A) 130.2(6) C(10A)—N(3A)—C(16A) 101.3(4)C(15A)—N(3A)—C(16′) 102.0(5) C(10A)—N(3A)—C(16′) 119.1(4)C(16A)—N(3A)—C(16′) 28.2(3) C(15A)—N(3A)—H(3A) 103.9 C(10A)—N(3A)—H(3A)103.9 C(16A)—N(3A)—H(3A) 103.9 C(16′)—N(3A)—H(3A) 116.0C(6B)—N(1B)—C(7B) 125.8(3) C(6B)—N(1B)—H(1B) 117.1 C(7B)—N(1B)—H(1B)117.1 C(3B)—N(2B)—C(13B) 114.9(2) C(3B)—N(2B)—C(14B) 109.1(2)C(13B)—N(2B)—C(14B) 111.1(3) C(3B)—N(2B)—H(2B1) 107.1C(13B)—N(2B)—H(2B1) 107.1 C(14B)—N(2B)—H(2B1) 107.1 C(10B)—N(3B)—C(15B)114.9(2) C(10B)—N(3B)—C(16B) 110.6(2) C(15B)—N(3B)—C(16B) 110.8(2)C(10B)—N(3B)—H(3B) 106.7 C(15B)—N(3B)—H(3B) 106.7 C(16B)—N(3B)—H(3B)106.7 O(1S′)—S(1)—O(2S) 131.2(3) O(1S′)—S(1)—O(3S′) 123.5(3)O(2S)—S(1)—O(3S) 25.1(2) O(1S′)—S(1)—O(3S) 71.7(3) O(2S)—S(1)—O(3S)110.8(3) O(3S′)—S(1)—O(3S) 134.9(3) O(1S′)—S(1)—O(2S′) 116.5(3)O(2S)—S(1)—O(2S′) 84.3(3) O(3S′)—S(1)—O(2S′) 107.5(3) O(3S)—S(1)—O(2S′)45.0(2) O(1S′)—S(1)—O(1S) 33.5(2) O(2S)—S(1)—O(1S) 109.1(3)O(3S′)—S(1)—O(1S) 93.0(3) O(3S)—S(1)—O(1S) 103.3(3) O(2S′)—S(1)—O(1S)148.0(3) O(1S′)—S(1)—C(1S) 107.0(2) O(2S)—S(1)—C(1S) 114.7(2)O(3S′)—S(1)—C(1S) 102.3(2) O(3S)—S(1)—C(1S) 113.6(2) O(2S′)—S(1)—C(1S)95.2(2) O(1S)—S(1)—C(1S) 104.4(2) O(4S)—S(2)—O(5S) 112.76(15)O(4S)—S(2)—O(6S) 111.68(16) O(5S)—S(2)—O(6S) 112.07(17) O(4S)—S(2)—C(2S)108.24(17) O(5S)—S(2)—C(2S) 106.71(17) O(6S)—S(2)—C(2S) 104.86(16)O(9S)—S(3)—O(7S) 112.41(17) O(9S)—S(3)—O(8S) 113.91(16) O(7S)—S(3)—O(85)111.12(14) O(9S)—S(3)—C(3S) 105.99(19) O(7S)—S(3)—C(3S) 107.38(18)O(8S)—S(3)—C(3S) 105.42(16) O(11S)—S(4)—O(12S) 112.8(2)O(11S)—S(4)—O(10S) 114.1(2) O(12S)—S(4)—O(10S) 110.8(2)O(11S)—S(4)—C(4S) 106.2(3) O(12S)—S(4)—C(4S) 107.6(2) O(10S)—S(4)—C(4S)104.6(2) C(1A)—S(1A)—C(12A) 100.76(19) C(12B)—S(1B)—C(1B) 101.93(14)Symmetry transformations used to generate equivalent atoms:

TABLE 4 Anisotropic displacement parameters (Å² × 10³) for eul1_0m. Theanisotropic displacement factor exponent takes the form: −2π²[h²a*²U¹¹ + . . . + 2 h k a* b* U¹²] U¹¹ U²² U³³ U²³ U¹³ U¹² C(1A) 22(1)45(2) 19(1) 2(1) 1(1) 21(1) C(2A) 17(1) 41(2) 20(1) 4(1) 0(1) 14(1)C(3A) 19(1) 46(2) 15(1) 2(1) 1(1) 12(1) C(4A) 19(1) 57(2) 19(1) −4(1) 2(1) 12(1) C(5A) 19(1) 66(2) 20(1) −6(1)  −2(1)  21(1) C(6A) 23(1) 59(2)17(1) −4(1)  −2(1)  26(1) C(7A) 75(2) 90(2) 15(1) 8(2) 5(1) 73(2) C(8A)86(2) 98(2) 19(1) 6(2) 0(1) 83(2) C(9A) 101(2)  92(2) 20(2) 6(2) −2(2) 86(2)  C(10A) 100(2)  76(2) 18(1) 9(1) 3(2) 78(2)  C(11A) 86(2) 71(2)16(1) 14(1)  7(1) 69(2)  C(12A) 75(2) 75(2) 15(1) 15(1)  8(1) 67(2) C(13A) 27(2) 44(2) 20(2) 8(2) 4(2) −3(2)  C(14A) 39(2) 30(2) 25(2) 7(2)10(2)   5(2)  C(15A) 104(4)  34(2) 28(2) 4(2) −10(2)  34(3) C(1B) 17(1)22(1) 18(1) 4(1) 3(1) 10(1) C(2B) 21(1) 22(1) 18(1) 6(1) 4(1) 10(1)C(3B) 19(1) 23(1) 19(1) 5(1) 4(1) 10(1) C(4B) 20(1) 23(1) 22(1) 6(1)3(1) 12(1) C(5B) 22(1) 21(1) 24(1) 7(1) 4(1) 11(1) C(6B) 18(1) 22(1)23(1) 5(1) 6(1) 10(1) C(7B) 21(1) 20(1) 23(1) 6(1) 6(1) 11(1) C(8B)23(1) 19(1) 26(1) 6(1) 6(1) 11(1) C(9B) 21(1) 19(1) 26(1) 5(1) 7(0  9(1)  C(10B) 19(1) 21(1) 19(1) 3(1) 4(1) 10(1)  C(11B) 20(1) 21(1)20(1) 5(1) 5(1) 10(1)  C(12B) 18(1) 20(1) 20(1) 6(1) 5(1)  8(1)  C(13B)21(2) 30(2) 24(2) 10(2)  4(1)  7(1)  C(14B) 24(2) 26(2) 21(2) 0(1) 0(1) 6(1)  C(15B) 24(2) 22(2) 25(2) 0(1) 0(1)  8(1)  C(16B) 27(2) 31(2)27(2) 0(2) 2(1) 18(2) C(1S)  33(2) 49(2) 28(2) 7(2) 0(2) 20(2) C(2S) 35(2) 29(2) 26(2) 2(2) −6(2)  12(2) C(3S)  62(3) 44(2) 31(2) 12(2)  9(2)40(2) C(4S)  95(4) 51(3) 30(2) 16(2)  22(2)  11(3) N(1A) 42(2) 88(3)23(2) 2(2) −6(1)  52(2) N(2A) 20(1) 32(2) 19(1) 5(1) 2(1)  0(1) N(3A)159(4)  58(2) 17(2) 1(2) −12(2)  84(3) N(1B) 18(1) 25(1) 34(2) 17(1) 7(1)  9(1) N(2B) 17(1) 25(1) 21(1) 6(1) 2(1) 10(1) N(3B) 18(1) 20(1)21(1) 2(1) 1(1)  8(1) O(4S)  39(2) 25(1) 33(1) 10(1)  12(1)   9(1)O(5S)  37(2) 35(2) 34(2) 0(1) 14(1)  10(1) O(6S)  30(1) 27(1) 54(2)14(1)  −11(1)   8(1) O(7S)  27(1) 48(2) 42(2) 28(1)  16(1)  22(1) O(8S) 33(1) 42(1) 34(1) 23(1)  13(1)  25(1) O(9S)  33(2) 28(1) 53(2) 7(1)−5(1)   3(1)  O(10S) 96(2) 27(1) 26(1) 4(1) −11(2)  24(2)  O(11S) 84(3)128(3)  45(2) 25(2)  15(2)  82(3)  O(12S) 43(2) 54(2) 28(2) 2(1) −5(1) −11(1)  S(1)  21(1) 19(1) 45(1) 13(1)  −12(1)   5(1) S(2)  23(1) 22(1)20(1) 5(1) 3(1)  9(1) S(3)  19(1) 22(1) 28(1) 10(1)  6(1) 11(1) S(4) 29(1) 22(1) 18(1) 6(1) 2(1)  4(1)  S(1A) 29(1) 43(1) 39(1) 19(1)  5(1)26(1)  S(1B) 17(1) 22(1) 39(1) 15(1)  1(1)  7(1)

TABLE 5 Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for eul1_0m. x y z U(eq) H(2A) 1662 7847 2452 32H(4A) 5495 10304 2671 45 H(5A) 6007 9108 3318 47 H(8A) 5053 5489 4110 66H(9A) 3662 3536 4304 69 H(11A) 536 3874 3547 54 H(13A) 4800 11131 182758 H(13B) 3517 10752 1260 58 H(13C) 4057 9728 1211 58 H(14A) 2513 106273064 54 H(14B) 2623 11370 2445 54 H(14C) 3910 11643 2966 54 H(15A) 11733266 5213 84 H(15B) −13 1828 4928 84 H(15C) −105 2958 4669 84 H(16H) −60541 3357 32 H(16I) 1086 1487 3019 32 H(16J) −179 1679 3122 32 H(16K)−683 569 3701 56 H(16L) 94 977 3039 56 H(16M) −708 1698 3407 56 H(2B)301 2081 1708 24 H(4B) −1035 4090 775 25 H(5B) 1095 5709 911 26 H(8B)5567 7602 1559 27 H(9B) 7524 7850 2158 27 H(11B) 5531 4439 2601 24H(13D) −1727 843 1744 41 H(13E) −3189 582 1529 41 H(13F) −2161 1901 214841 H(14D) −1922 1307 −87 41 H(14E) −2973 184 197 41 H(14F) −1468 547 35541 H(15D) 9282 8071 2855 39 H(15E) 9634 7519 3485 39 H(15F) 8555 80043570 39 H(16A) 7739 4713 1898 43 H(16B) 9145 5506 2426 43 H(16C) 87526089 1829 43 H(1S1) 4324 513 5083 56 H(1S2) 3770 163 4204 56 H(1S3) 3106−863 4655 56 H(2S1) 8396 8376 210 50 H(2S2) 8124 7229 571 50 H(2S3) 92377616 64 50 H(3S1) 5170 3997 −144 60 H(3S2) 6146 4646 652 60 H(3S3) 46613623 594 60 H(4S1) 6520 2129 4255 100 H(4S2) 5930 3121 4436 100 H(4S3)7389 3569 4811 100 H(1A) 5282 7506 3975 55 H(2A1) 2289 9350 1848 35H(3A) 1596 2011 4279 82 H(1B) 3327 6282 1083 29 H(2B1) −2456 2212 994 26H(3B) 7679 5766 3119 25References

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The invention claimed is:
 1. A method of treating a tauopathy comprisingadministering to a subject in need thereof the compound of the followingformula:

or a pharmaceutically acceptable salt, solvate, or hydrate thereof. 2.The method of claim 1, wherein the tauopathy is Alzheimer's disease(AD), Pick's disease, progressive supranuclear palsy (PSP),frontotemporal dementia (FTD), FTD with parkinsonism linked tochromosome 17 (FTDP 17), frontotemporal lobar degeneration (FTLD)syndromes; disinhibition-dementia-parkinsonism-amyotrophy complex(DDPAC), pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome,pallido nigro luysian degeneration (PNLD), cortico-basal degeneration(CBD), dementia with argyrophilic grains (AgD), dementia pugilistica(DP) or chronic traumatic encephalopathy (CTE), Down's syndrome (DS),dementia with Lewy bodies (DLB), subacute sclerosing panencephalitis(SSPE), Niemann-Pick disease, type C (NPC), Sanfilippo syndrome type B,mucopolysaccharidosis III B (MPS III B)), or myotonic dystrophies (DM),DM1 or DM2.
 3. The method of claim 2, wherein the tauopathy isAlzheimer's disease.
 4. The method of claim 2, wherein the tauopathy isPSP.
 5. The method of claim 2, wherein the tauopathy is FTD, FTDP-17, orFTLD.